Antibodies and methods of use

ABSTRACT

The presently disclosed subject matter provides antibodies that bind KLB and FGFR1, and methods of using the same. In certain embodiments, an antibody of the present disclosure includes a bispecific antibody that binds to an epitope present on FGFR1 and binds to an epitope present on KLB.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.17/341,990, filed Jun. 8, 2021, which is a continuation of U.S. patentapplication Ser. No. 17/131,980, filed Dec. 23, 2020, now abandoned,which is a divisional of U.S. patent application Ser. No. 16/284,774,filed Feb. 25, 2019, now U.S. Pat. No. 10,882,921, which is a divisionalof U.S. patent application Ser. No. 15/837,801, filed Dec. 11, 2017, nowU.S. Pat. No. 10,246,518, which is a divisional of U.S. patentapplication Ser. No. 14/582,100, filed Dec. 23, 2014, now U.S. Pat. No.9,873,748, which claims priority to U.S. Provisional Patent ApplicationSer. No. 61/920,396, filed Dec. 23, 2013, and U.S. Provisional PatentApplication Ser. No. 62/081,435, filed Nov. 18, 2014, and the contentsof each of the above-listed applications are incorporated by referenceherein in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Mar. 31, 2023, isnamed 00B206_1351.xml and is 221,933 bytes in size.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to beta-Klotho(KLB), Fibroblast Growth Factor Receptor 1 (FGFR1), or both, and methodsof using the same.

BACKGROUND

Fibroblast growth factor 21 (FGF21) and its closest homologue FGF19 aremembers of the FGF superfamily. FGF21 signaling requires FGF-receptor(FGFR) isoforms and the membrane-bound coreceptor Klotho-beta (KLB)(Ogawa et al. Proc. Natl. Acad. Sci. USA 104(18): 7432-37 (2007);US2010/0184665). FGF19 has also been shown to signal through FGFRisoforms complexed with KLB (Wu et al. J. Biol. Chem. 282(40):29069-29072 (2007)). Of the 7 primary isoforms of FGFR encoded bymammalian species (1b, 2b, 3b, 1c, 2c, 3c, and 4), only three isoforms,FGFR1c, 2c and 3c, can transduce signaling by both FGF19 and FGF21 whenbound by coreceptor KLB, which is predominantly expressed in the liver,adipose tissue, and pancreas (Goetz and Mohammadi, Nature reviews.Molecular Cell Biology 14, 166-180 (2013)). Of these receptors, FGFR1cappears to play a predominant role in mediating the metabolic effect ofFGF21. Without being bound to a particular theory, it is believed thatFGF21 acts by inducing homodimerization of FGFR isoforms in the presenceof the membrane-bound co-receptor KLB. Unlike other FGF ligands, FGF21exhibits very low affinity to any individual FGFR. However, highaffinity binding to KLB through the C-terminal tail region recruitsFGF21 to the FGFR/KLB complex, allowing FGF21 to interact with FGFRsdespite the low affinity to FGFRs alone.

FGF21 was identified as a potent disease-modifying protein agent toreverse obesity and type 2 diabetes in animal disease models(Kharitonenkov et al. J Clin. Invest. 115(6): 1627-35 (2005)).Recombinant FGF21 has been shown to reduce hepatic lipids, improveinsulin sensitivity, and normalize glycemic control inleptin-signaling-deficient (ob/ob or db/db) mice or high-fat diet(HFD)-fed mice. Reduction in blood glucose and improvements in variouscardiovascular risk factors have also been observed in obese anddiabetic rhesus monkeys treated daily with recombinant FGF21. FGF21 andFGF19 have both been shown to activate the thermogenic function ofuncoupling protein 1 (UCP1)-positive adipose tissues (brown and beigeadipose tissues; BAT) in obese rodents (Fu et al., Endocrinology 145,2594-2603 (2004); Coskun et al., Endocrinology 149, 6018-6027 (2008);Fisher et al., Genes & Development 26, 271-281 (2012)).

Although clinical applications of recombinant FGF21 or FGF19 analogs arecurrently being tested for the treatment of metabolic disease, theirdevelopment for therapeutic intervention has proven challenging. Forexample, the serum half-life of FGF21, ˜2 hours in non-human primates,is too short for practical clinical application and the remaining FGF21protein in circulation can be rapidly inactivated by proteolyticcleavage. Efforts have been made to improve these properties throughprotein engineering, but such modifications could increaseimmunogenicity and other modification-specific adverse effects. Anothersignificant challenge is a possibility of long-term adverse effects fromchronic FGF21-mediated therapy. For example, FGF21 has been reported toinduce hepatic growth hormone resistance via induction of SOCS2, aninhibitor of growth hormone receptor signaling (Inagaki et al., CellMetab. 8: 77-83 (2008)). In humans, growth hormone resistance ordeficiency is associated with low bone mass in children and adults andtransgenic overexpression of FGF21 or two weeks treatment of mice withrecombinant FGF21 leads to a dramatic loss of bone mineral density. Ithas not yet been demonstrated that the bone-related adverse effects ofFGF21 can be de-linked from the beneficial metabolic effects. Further,transgenic overproduction of FGF19 can lead to hepatocellularcarcinogenesis via activation of FGF Receptor (FGFR) 4 (Fu et al.,Endocrinology 145, 2594-2603 (2004); Tomlinson et al., Endocrinology143, 1741-1747 (2002); French et al., PLoS One 7, e36713 (2012)).

Recombinant monoclonal antibodies (Abs) can act as a powerfultherapeutic modality as they can provide excellent target selectivity,pharmacokinetic profile, and other properties important for apharmaceutical agent (Chan and Carter, Nature reviews. Immunology 10,301-316 (2010)). For example, an antibody antagonist specific for FGFR1cwas reported to induce weight loss in mice and monkeys (WO2005/037235)and agonistic antibody-mediated selective activation of FGFR1c issufficient to recapitulate the insulin sensitization by FGF21 indiabetic mice (WO2012/158704; Wu et al. Science Translational Med.3(113): 1-10 (2011)). Antibodies that bind to the KLB/FGFR1c complexhave been proposed as activators/therapeutic agents (U.S. Pat. No.7,537,903; WO2011/071783; WO2012/158704). Others have investigated twoalternative approaches to selectively activate the FGFR1c/KLB complex,such as a high affinity anti-KLB antibody called mimAb1 (Foltz et al.Sci. Transl. Med. 4: 162ra153 (2012)) and bispecific anti-FGFR1/KLBAvimer polypeptide C3201 linked to human serum albumin (HSA) (U.S. Pat.No. 8,372,952).

Given the significant role for FGF19 and FGF21 in glucose metabolism,there remains a need in the art for the development of therapeuticmolecules and methods to modulate FGF19 or FGF21-mediated activities.

SUMMARY

The present disclosure provides antibodies that bind to KLB, antibodiesthat bind to FGFR1, and bispecific antibodies that bind to both KLB andFGFR1, and methods of using the same. The invention is based, in part,on the discovery of bispecific antibodies that bind to both KLB andFGFR1, which selectively activate the FGFR1c/KLB receptor complex andinduce the beneficial metabolic changes expected from the FGF21-likeactivity, including weight loss and improvement in glucose and lipidmetabolism, without a significant impact on the liver and without a lossin bone mass.

In certain embodiments, the antibody is a bispecific antibody. Forexample, and not by way of limitation, an isolated antibody of thepresent invention can bind to both beta-Klotho (KLB) and FibroblastGrowth Factor Receptor 1 (FGFR1), wherein the antibody binds to theC-terminal domain of KLB. For example and not by way of limitation, anisolated antibody of the present disclosure binds to both KLB andFGFR1c. In certain embodiments, the antibody binds to a fragment of KLBincluding the amino acid sequence SSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS(SEQ ID NO: 142). In certain embodiments, the antibody binds to anepitope within a fragment of FGFR1 including the amino acid sequenceKLHAVPAAKTVKFKCP (SEQ ID NO: 143) or FKPDHRIGGYKVRY (SEQ ID NO: 144).

In certain embodiments, an antibody of the present disclosure activatesthe KLB/FGFR1c complex. In certain embodiments, an antibody of thepresent disclosure reduces blood glucose levels in vivo. In certainembodiments, the antibody does not significantly affect bone density. Incertain embodiments, an antibody of the present disclosure does not havea significant impact on the liver. In certain embodiments, the antibodyinduces ERK and MEK phosphorylation in the liver at significantly lowerlevels than FGF21 induces. In certain embodiments, the antibody binds toKLB with a K_(d) from 10⁻⁸ M to 10⁻¹³ M. In certain embodiments, anantibody of the present disclosure can bind to a FGFR1 protein with aK_(d) from 10⁻⁸ M to 10⁻¹³ M. In certain embodiments, an antibody of thepresent disclosure can bind to FGFR1c with a K_(d) from 10⁻⁸ M to 10⁻¹³M. In certain embodiments, the antibody binds to KLB with a K_(d) of <10nM and to FGFR1c with a K_(d) of >300 nM. In certain embodiments, ananti-KLB/anti-FGFR1 bispecific antibody can include an anti-FGFR1 armthat has a K_(d) of about 10 nM to about 10 μM.

In certain embodiments, an antibody of the present disclosure binds toan epitope present on KLB. For example, and not by way of limitation,the present disclosure provides an anti-KLB antibody that binds the sameepitope as an antibody shown in FIGS. 3A and B. In certain embodiments,an anti-KLB antibody of the present disclosure binds the same epitope asthe 12A11 or the 8C5 antibody. In certain embodiments, the anti-KLBantibody binds to an epitope within the C-terminal domain of KLB. Incertain embodiments, the anti-KLB antibody binds to a fragment of KLBconsisting of the amino acid sequence SSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS(SEQ ID NO: 142).

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody ofthe present disclosure includes a first antibody, or antigen bindingportion thereof, that includes a heavy chain variable region and a lightchain variable region, where the heavy chain variable region includesamino acids having a sequence that is at least 95% identical to thesequence set forth in SEQ ID NO: 128, and the light chain variableregion includes amino acids having a sequence that is at least 95%identical to the sequence set forth in SEQ ID NO: 130. In certainembodiments, the second antibody, or antigen binding portion thereof,includes a heavy chain variable region and a light chain variableregion, where the heavy chain variable region includes amino acidshaving a sequence that is at least 95% identical to the sequence setforth in SEQ ID NO: 132, and the light chain variable region includesamino acids having a sequence that is at least 95% identical to thesequence set forth in SEQ ID NO: 134.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody ofthe present disclosure includes a first antibody, or antigen bindingportion thereof, which includes a heavy chain region and a light chainregion, where the heavy chain region includes amino acids having asequence that is at least 95% identical to the sequence set forth in SEQID NO: 129, and the light chain region includes amino acids having asequence that is at least 95% identical to the sequence set forth in SEQID NO: 131. In certain embodiments, the second antibody, or antigenbinding portion thereof, includes a heavy chain region and a light chainregion, where the heavy chain region includes amino acids having asequence that is at least 95% identical to the sequence set forth in SEQID NO: 133, and the light chain region includes amino acids having asequence that is at least 95% identical to the sequence set forth in SEQID NO: 135.

The present disclosure further provides an anti-KLB antibody thatincludes: (a) HVR-H3 comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1-15, (b) HVR-L3 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 79-93,and (c) HVR-H2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 16-31.

In certain embodiments, the anti-KLB antibody comprises (a) HVR-H1comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1-15, (b) HVR-H2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 16-31, and (c) HVR-H3comprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 32-47.

In certain embodiments, the anti-KLB antibody further comprises (a)HVR-L1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 48-62, (b) HVR-L2 comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 63-78, and(c) HVR-L3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 79-93.

In certain embodiments, the anti-KLB antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 12, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 28, (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 44, (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 59, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 75, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 90.

In certain embodiments, the anti-KLB antibody comprises (a) HVR-H1comprising the amino acid sequence of SEQ ID NO: 15, (b) HVR-H2comprising the amino acid sequence of SEQ ID NO: 31, (c) HVR-H3comprising the amino acid sequence of SEQ ID NO: 47, (d) HVR-L1comprising the amino acid sequence of SEQ ID NO: 62, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 78, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 93.

In certain embodiments, the anti-KLB antibody comprises (a) a heavychain variable region comprising the amino acid sequence of SEQ ID NO:128 and (b) a light chain variable region comprising the amino acidsequence of SEQ ID NO: 130. In certain embodiments, the antibodycomprises (a) a heavy chain comprising the amino acid sequence of SEQ IDNO: 129 and (b) a light chain comprising the amino acid sequence of SEQID NO: 131.

In another aspect, the present disclosure provides an anti-KLB antibodycomprising (a) a heavy chain variable region having at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 128; (b) alight chain variable region having at least 95% sequence identity to theamino acid sequence of SEQ ID NO: 130; and (c) a heavy chain variableregion as in (a) and a light chain variable region as in (b).

The present disclosure further provides antibodies that bind to FGFR1,e.g., FGFR1c. For example, and not by way of limitation, an antibody ofthe present disclosure comprises a variable domain that binds to FGFR1.In certain embodiments, the antibody binds to a fragment of FGFR1consisting of the amino acid sequence KLHAVPAAKTVKFKCP (SEQ ID NO: 143)or FKPDHRIGGYKVRY (SEQ ID NO: 144). In certain embodiments, the antibodycomprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:136, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 137,(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 138, (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 140, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO: 141. In certainembodiments, the antibody comprises (a) a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 132 and (b) a lightchain variable region comprising the amino acid sequence of SEQ ID NO:134. In certain embodiments, the antibody comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 133 and (b) a lightchain comprising the amino acid sequence of SEQ ID NO: 135. In certainembodiments, an antibody of the present disclosure binds to a fragmentof FGFR1c consisting of the amino acid sequence KLHAVPAAKTVKFKCP (SEQ IDNO: 143) or FKPDHRIGGYKVRY (SEQ ID NO: 144).

In certain embodiments, an antibody of the present disclosure is amonoclonal antibody. In certain embodiments, the antibody is a human,humanized, or chimeric antibody. In certain embodiments, the antibodyhas reduced effector function.

In another aspect, the present disclosure provides an isolated nucleicacid encoding an antibody of the present disclosure. In certainembodiments, the present disclosure provides a host cell comprising anucleic acid of the present disclosure. In certain embodiments, thepresent disclosure provides a method of producing an antibody comprisingculturing a host cell of the present disclosure so that the antibody isproduced. In certain embodiments, this method further comprisesrecovering the antibody from the host cell.

The present disclosure further provides a pharmaceutical formulationthat includes one or more antibodies of the invention and apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical formulation comprises an additional therapeutic agent.

In another aspect, the present disclosure provides an antibody of theinvention for use as a medicament. In certain embodiments, the antibodyis for use in treating metabolic disorders, e.g., polycystic ovarysyndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholicsteatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD),hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type1 diabetes, latent autoimmune diabetes (LAD), and maturity onsetdiabetes of the young (MODY). In certain embodiments, the antibody isfor use in treating type 2 diabetes. In certain embodiments, theantibody is for use in treating obesity. In certain embodiments, thepresent disclosure provides an antibody for use in treating Bardet-Biedlsyndrome, Prader-Willi syndrome, Alstrom syndrome, Cohen syndrome,Albright's hereditary osteodystrophy (pseudohypoparathyroidism),Carpenter syndrome, MOMO syndrome, Rubinstein-Taybi syndrome, fragile Xsyndrome and Börjeson-Forssman-Lehman syndrome. In certain embodiments,the present disclosure provides an antibody for use in activating aKLB/FGFR1 receptor complex, e.g., a KLB/FGFR1c receptor complex.

In another aspect, the present disclosure provides the use of anantibody, disclosed herein, in the manufacture of a medicament fortreatment of metabolic disorders, e.g., polycystic ovary syndrome(PCOS), metabolic syndrome (MetS), obesity, non-alcoholicsteatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD),hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type1 diabetes, latent autoimmune diabetes (LAD), and maturity onsetdiabetes of the young (MODY), and aging and related diseases such asAlzheimer's disease, Parkinson's disease and ALS. In certainembodiments, the metabolic disorder is type 2 diabetes. In certainembodiments, the metabolic disorder is obesity. In certain embodiments,the manufacture is of a medicament for activating a KLB/FGFR1c receptorcomplex.

In another aspect, the present disclosure provides a method of treatingan individual having a disease selected from the group consisting ofpolycystic ovary syndrome (PCOS), metabolic syndrome (MetS), obesity,non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease(NAFLD), hyperlipidemia, hypertension, type 2 diabetes, non-type 2diabetes, type 1 diabetes, latent autoimmune diabetes (LAD), andmaturity onset diabetes of the young (MODY), and aging and relateddiseases such as Alzheimer's disease, Parkinson's disease and ALS, themethod comprising administering to the individual an effective amount ofone or more antibodies of the present disclosure. In certainembodiments, the disease is diabetes, e.g., type 2 diabetes. In certainembodiments, the disease is obesity. In certain embodiments, the presentdisclosure provides a method of treating an individual having a diseaseand/or disorder selected from the group consisting of Bardet-Biedlsyndrome, Prader-Willi syndrome, Alstrom syndrome, Cohen syndrome,Albright's hereditary osteodystrophy (pseudohypoparathyroidism),Carpenter syndrome, MOMO syndrome, Rubinstein-Taybi syndrome, fragile Xsyndrome and Börjeson-Forssman-Lehman syndrome. In certain embodiments,the method further includes administering an additional therapeuticagent to the individual. In certain embodiments, a method using one ormore antibodies of the present disclosure does not affect liver functionin an individual. In certain embodiments, the present disclosureprovides a method for inducing weight loss comprising administering toan individual an effective amount of one or more antibodies of thepresent disclosure.

In another aspect, the present disclosure provides a method ofactivating a KLB-FGFR1c receptor complex in an individual comprisingadministering to the individual an effective amount of an antibody ofthe present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts agonistic activity of anti-FGFR1 antibodies and antibodyfragments.

FIG. 1B depicts results of binding competition experiments usinganti-FGFR1 antibodies.

FIG. 1C depicts amino acid residues in FGFR1 important for binding byanti-FGFR1 antibodies of the presently disclosed subject matter. FIG. 1Cdiscloses SEQ ID NOs: 159, 159, 143 and 144, respectively, in order ofappearance.

FIG. 1D depicts the results of site-specific mutagenesis to determineamino acid residues important for binding by anti-FGFR1 antibodies ofthe presently disclosed subject matter.

FIG. 1E depicts the results of site-specific mutagenesis to determineamino acid residues important for binding by anti-FGFR1 antibodies ofthe presently disclosed subject matter.

FIG. 1F depicts residues important for binding on a space-filling modelof FGFR1.

FIG. 2A depicts the affinities of two anti-FGFR1 antibodies for FGFR1band FGFR1c.

FIG. 2B depicts binding of an anti-FGFR1 antibody to various FGFRs.

FIG. 2C depicts an anti-FGFR1 antibody that acted as a specific agonistfor FGFR1 in GAL-ELK1 (ETS-like transcription factor 1) based luciferaseassay in L6 cells.

FIG. 2D depicts that an anti-FGFR1 antibody acted as a specific agonistfor FGFR1 in GAL-ELK1 based luciferase assay in HEK293 cells.

FIG. 2E depicts that an anti-FGFR1 antibody normalized blood glucoselevels when injected into diabetic ob/ob mice.

FIG. 3A depicts the light chain variable region sequences for 17anti-KLB antibodies. The CDR L1 sequences are, in order, SEQ ID NOs:48-62; the CDR L2 sequences are, in order, SEQ ID NOs: 63-78; and theCDH L3 sequences are, in order, SEQ ID NOs: 79-93. The light chainvariable region sequences are, in order, SEQ ID NOs: 111-127.

FIG. 3B depicts the heavy chain variable region sequences for 17anti-KLB antibodies. The CDR H1 sequences for the antibodies are, inorder (11F1-8C5), SEQ ID NOs: 1-15; the CDR H2 sequences are, in order,SEQ ID NOs: 16-31; the CDR H3 sequences are, in order, SEQ ID NOs:32-47. The heavy chain variable region sequences for the antibodies are,in order, SEQ ID NOs: 94-110.

FIG. 4 depicts the median shift observed in the FACS plot at 0.8 μg/mlmeasuring binding of various anti-KLB antibodies to 293 cells expressinghKLB.

FIG. 5 depicts the relative binding of various anti-KLB antibodies tohKLB-ECD-HIS protein.

FIG. 6A is a schematic diagram representing antibodies of the presentlydisclosed subject matter and a model for KLB/FGFR1c bispecific Abcomplex formation for signal activation.

FIG. 6B depicts a model for FGFR1c-KLB-FGF21 complex formation forsignal activation.

FIG. 6C depicts a GAL-ELK1 luciferase assay of FGF21 and bispecificantibody (BsAb) 17 activity using FGFR1-deficient HEK293 cells. Cellswere transfected to express indicated receptors.

FIG. 6D depicts a western blot analysis of primary human adipocytestreated with the indicated protein (FGF21 (100 nM) or IgG (33 nM)) for 1hr. Samples were duplicated for each treatment.

FIG. 7A depicts induction by various bispecific antibodies withanti-FGFR1 and anti-KLB arms in a GAL-ELK1 based luciferase assay. Notethat bispecific Abs with R1MAb1 arm exhibited significantKLB-independent activity, presumably due to the agonistic activity ofR1MAb1 Fab. No such activity was observed with bispecific Abs withR1MAb2 or R1MAb3 arm.

FIG. 7B depicts that induction of signaling by various bispecificantibodies with anti-FGFR1 and anti-KLB arms is dependent on both FGFR1cand KLB.

FIG. 7C depicts a bispecific antibody with anti-FGFR1 and anti-KLB armsthat induced luciferase activity in a dose-dependent manner in cellsexpressing recombinant hFGFR1c and hKLB, but not in cells without KLBexpression.

FIG. 8A is a schematic representation of three variants ofanti-KLB/anti-FGFR1c bispecific antibodies. Blue: human, and Red: mouse.Approximate position of the oligosaccharide chain at N297 in (1) isindicated by {circumflex over ( )}. The effector-less versions ((2) and(3)) lack the oligosaccharide chains due to the N297G mutation.Asterisks in (2) indicate approximate position of the D265A mutation.The orientation of knob vs hole is also shown. (1) represents BsAb10;(2) represents BsAb20; and (3) represents BsAb17.

FIG. 8B depicts an MSD pERK assay in primary human adipocytes treatedwith BsAb10 and its derivatives, control IgG or FGF21 for 10 min. Datarepresent means±SEM (N=3). bFKB1 (1) represents BsAb10; bFKB1 (2)represents BsAb20; and bFKB1 (3) represents BsAb17.

FIG. 9A depicts a GAL-ELK1 luciferase assay in rat L6 myoblast cells.Cells were co-transfected with an expression vector for indicatedreceptors. Transfected cells were incubated with various concentrationsof BsAb10 or a positive control, FGF21, FGF19 or R1MAb1, for 6 h beforeluciferase assays.

FIG. 9B depicts similar GAL-ELK1 luciferase assays as shown in FIG. 9A.Transfected L6 cells were treated with combinations of FGF21 and BsAb17as indicated. N=4.

FIG. 9C depicts similar GAL-ELK1 luciferase assays as shown in FIG. 9A.Transfected L6 cells were treated with combinations of FGF21 and BsAb17as indicated. N=4.

FIG. 9D depicts the binding of an anti-FGFR1 antibody and theanti-KLB/anti-FGFR1 bispecific antibodies, BsAB9 and BsAb10, to cellsexpressing KLB, FGFR1c or both.

FIG. 10A depicts binding of a bispecific antibody with anti-FGFR1 andanti-KLB arms and an anti-FGFR1 antibody to cells expressing FGFR1c, KLBor both.

FIG. 10B depicts the K_(d) of BsAb10 for binding to HEK293 cellexpressing various combinations of human and murine KLB/FGFR1.

FIG. 11 depicts the binding analysis of BsAb10 or preformed BsAb10/KLBcomplexes at 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM toFGFR1-ECD-Fc fusion protein that was immobilized on the chip. Togenerate preformed BsAb10/KLB complexes, BsAb10 and recombinant KLB-ECDprotein was preincubated at 1:1 ratio. Note the dissociation rate wasslower with BsAb10/KLB complex than with BsAb10 alone, but only whenFGFR1c, but not FGFR1b, was captured on the chip, indicating theformation of a ternary complex.

FIG. 12A is a schematic representation of the TR-FRET experiment design.

FIG. 12B depicts the TR-FRET intensity on COS7 cells expressing labeledSNAP-tagged FGFR1c protein with or without untagged KLB at 15 minutesafter addition of indicated ligands. BsAb17, FGF21, FGF1 and FGF2 wereused at 67 nM, 50 nM, 62.5 nM, 12 nM, respectively. The data representsFRET intensity at 665 nm divided by the donor emission at 620 nm (FRETratio), and means±SEM of three independent experiments (N=3). p<0.05(*), <0.01 (**), <0.0001 (***) vs PBS control.

FIG. 13A depicts the results of experiments to determine which part ofKLB was important for binding by two anti-KLB antibodies. A schematicrepresentation of KLB protein structure is shown at the top. Each barrepresents human KLB, human KLA, rabbit KLB, rat KLB, mouse KLB, orchimeric constructs as color coded. At right, binding of KLBmAb1 andcontrol KLBmAb2 based on FACS with HEK293 cells transiently expressingeach construct is shown. Note that KLBmAb1 does not bind to rabbit KLB,but replacement of a 34 amino acid fragment (amino acid 805-838) to thecorresponding human sequence confers binding.

FIG. 13B depicts the amino acid sequence of the position 857-890 segmentof a human KLB protein with a signal sequence (which corresponds to theamino acid sequence at positions 805-838 of a KLB protein that does notinclude a signal sequence) and corresponding sequences in variousindicated species. FIG. 13B discloses SEQ ID NOs: 160-164, respectively,in order of appearance.

FIG. 14A depicts the binding of FGF21 and FGF19 to BsAb10/KLB complex bySPR. BsAb10 was captured on the chip, and KLB-ECD protein and FGFprotein (at 0.2, 0.8, or 2 μM) were sequentially injected.

FIG. 14B depicts the results of a GAL-ELK1 luciferase assay in rat L6myoblast cells. Cells were co-transfected with an expression vector forboth FGFR4 and KLB. Transfected cells were incubated with variousconcentrations of indicated proteins for 6 h before luciferase assays.

FIG. 14C depicts a Western blot that was performed to monitor ERKphosphorylation in H4IIE hepatoma cells. Note that BsAb17 did not blockthe ability of FGF19 to activate FGFR4/KLB complex (FIG. 14B), or toinduce ERK phosphorylation in H4IIE hepatoma cells (FIG. 14C).

FIG. 15A depicts the blood glucose levels (day 7), % body weight change(day 7) and daily food intake (day 0-3) of lean C57BL/6 and db/db mice(n=7) after a single intraperitoneal (i.p.) injection of BsAb17 orcontrol IgG at 3 mg/kg (lean) or 5 mg/kg (db/db).

FIG. 15B depicts the body weight and blood glucose levels of DietInduced Obesity (DIO) mice, which received i.p. injections of theindicated IgG (BsAb20) at 3 mg/kg on day 0 and 6 (arrows). N=9.

FIG. 15C depicts the results of the glucose tolerance test with the samemice and antibody used in 15B on day 14.

FIG. 15D depicts the amount of hepatic triglycerides, and serum markersin animals shown in 15B-C on day 17.

FIG. 15E depicts whole body glucose utilization, measured duringhyperinsulinaemic-euglycaemic clamps with DIO mice 5 days after a singlei.p. injection of the indicated IgG (BsAb17) at 10 mg/kg (N=12). Thehorizontal axis represents serum insulin levels. The arrows indicate thedirection of changes from basal to insulin-stimulated states. p<0.05(*), <0.005 (**), <0.0001 (***) vs control.

FIG. 15F depicts endogenous glucose production, measured duringhyperinsulinaemic-euglycaemic clamps with DIO mice 5 days after a singlei.p. injection of the indicated IgG (BsAb17) at 10 mg/kg (N=12). Thehorizontal axis represents serum insulin levels. The arrows indicate thedirection of changes from basal to insulin-stimulated states. p<0.05(*), <0.005 (**), <0.0001 (***) vs control.

FIG. 15G depicts insulin-stimulated tissue glucose uptake, measuredduring hyperinsulinaemic-euglycaemic clamps with DIO mice 5 days after asingle i.p. injection of the indicated IgG (BsAb17) at 10 mg/kg (N=12).p<0.05 (*), <0.005 (**), <0.0001 (***) vs control.

FIG. 16A shows the N-terminal amino acid sequence of mouse KLB protein(SEQ ID NO: 165), and the corresponding amino acid sequence encoded bythe Klb allele in the KO mice (SEQ ID NO: 166) are shown. A missensemutation in Klb gene results in a frame-shift after the second aminoacid in the KO allele, as shown with red letters.

FIG. 16B shows KLB protein expression in epididymal white adipose tissuein wildtype (+/+) and KLB knockout (−/−) mice.

FIG. 16C shows that KLB is important for BsAb20 to affect glucosemetabolism. Glucose tolerance test (GTT) in DIO mice that received fourweekly injections of BsAb20 or control IgG at 3 mpk. GTT was conductedon day 23, three days after the last injection. The mice were on HFD for20 weeks prior to GTT. *p<0.05.

FIG. 16D depicts the serum parameters in DIO mice on day 7 after an i.p.injection of an anti-KLB/anti-FGFR1 bispecific antibody or R1MAb1 at 50mg/kg or vehicle. N=6.

FIG. 17 depicts the amount of FGF23 and inorganic phosphorous in theserum of DIO mice on day 7 after i.p. injection of BsAb17 at 50 mg/kg.N=6. ***p<0.0005.

FIG. 18A depicts the amount of arterial blood glucose excursion duringthe clamp experiment. DIO mice received BsAb17 or control IgG at 10mg/kg on 5 days before the clamp experiment.

FIG. 18B depicts the body weight on the day of the clamp experiment.

FIG. 18C depicts the glucose infusion rate during the clamp experiment.p<0.05 (*), <0.001 (**) vs control.

FIG. 19A depicts the energy expenditure (EE) (left) and Respiratoryquotient (RQ) (right) of DIO mice that received a single i.p. injectionof 10 mg/kg IgG at the indicated time at 21-22° C. N=7.

FIG. 19B depicts the EE (top) and RQ (bottom) of lean mice that receiveda single i.p. injection of 10 mg/kg IgG at the indicated time. Mice weremaintained at 21-22° C., then cage temperature was shifted tothermoneutrality (29-30° C.) on 6 days post IgG injection. N=6-7.

FIG. 19C depicts the tissue fludeoxyglucose (FDG) uptake in DIO mice at40 hr after single i.p. injection of indicated IgG at 10 mg/kg. N=8.Mice were overnight fasted before FDG-uptake was measured.

FIG. 19D depicts the Western blot analysis of ingWAT harvested on day 7after single i.p. injection (BsAb17 or control IgG at 10 mg/kg) andsurgical implantation of an osmotic pump (CL316,243 (0.75 nmol/h) orvehicle).

FIG. 19E depicts the expression of Ucp1 mRNA in primary humansubcutaneous adipocytes treated with indicated protein at 30 nM for 48hr. N=3.

FIG. 19F depicts the core body temperature of DIO mice that received 10mg/kg of BsAb17 or control IgG. N=7-8.

FIG. 19G depicts the gene expression profile in iBAT of DIO micereceived single 10 mg/kg of IgG and FGF21 b.i.d. at 2 mg/kg/day orcontrol PBS for 5 days. All the genes that were significantly differentbetween BsAb17 and control, or between FGF21 and control were listed.

FIG. 19H depicts the EE (left) and RQ (right) of lean mice that receiveda single i.p. injection of an anti-KLB/anti-FGFR1 bispecific antibody orcontrol IgG at 10 mg/kg and surgical implantation of an osmotic pump(CL-316,243 at 0.5 nmol/h or vehicle) on day 0. The mean values duringthe indicated 24 h period are shown.

FIG. 20A depicts the amount of VO₂ (top), VCO₂ (middle) and totalactivity counts of DIO mice described in FIG. 19A. VO₂ and VCO₂ valuesare normalized by body weight values measured at times indicated by #.DIO mice received 10 mg/kg of BsAb17 or control IgG.

FIG. 20B depicts the amount of VO₂ (top), VCO₂ (middle) and totalactivity counts of DIO mice described in FIG. 19B. VO₂ and VCO₂ valuesare normalized by body weight values measured at times indicated by #.DIO mice received 10 mg/kg of BsAb17 or control IgG.

FIG. 21A depicts the average EE value in indirect calorimetry. Themagnitude in average increase is shown under the graphs. DIO 21° C.:Average value of EE during D3-D6 post IgG injection in the experimentshown in FIG. 19A. Lean 21° C.: Average value of EE during D3-D6 postIgG injection in the experiment shown in FIG. 19B. Lean after switch to30° C.: Average values of EE during D6-D9 post IgG injection (i.e., 3days after temperature switch) in the experiment shown in FIG. 19B. DIO30° C.: Average value of EE during D3-D6 post IgG injection in DIO miceacclimated at thermoneutrality.

FIG. 21B depicts the changes in EE in DIO mice at thermoneutrality. DIOmice were acclimated to thermoneutrality for 2 weeks prior to singlei.p. injection (arrow) of BsAb17 or control IgG at 10 mg/kg. N=3˜4.

FIG. 21C depicts the average EE and RQ in DIO mice at normal labtemperature (21° C.) during D3-5 after surgical implantation of anosmotic pump and drug injection. On DO, mice received i.p. injection ofBsAb17 or control IgG at 10 mg/kg. The FGF21 group also received bolus 2mg/kg FGF21 i.p. injection on DO. Each mouse was also subcutaneouslyimplanted with an osmotic pump to infuse FGF21 at 60 μg/day or PBScontrol on DO. N=8-9. ** p<0.005.

FIG. 22 depicts the data shown in FIG. 19F replotted to show the fitteddifference in core body temperature over the course of the study betweenDIO mice received 10 mg/kg of BsAb17 or control IgG. The black line isthe estimated difference and the blue lines are the 95% pointwiseconfidence intervals of the difference. IgG was administered at day 13(arrow). N=7-8.

FIG. 23 depicts the FGF21 and BsAb20-induced ERK and MEK phosphorylationto a similar extent in epididymal fat, inguinal fat, and interscapularbrown fat, and pancreas. Tissues were harvested at 1 h (liver, pancreasand epididymal white adipose tissue (eWAT)) or 2 h (iBAT or ingWAT)after i.p. injection of lean C57BL/6 mice at 10 mg/kg (BsAb20) or 1mg/kg (FGF21). Total ERK and MEK serve as loading controls.

FIG. 24A depicts the body weight changes and serum HMW adiponectinlevels in DIO mice (N=6) that received single i.p. of BsAb17 at theindicated dose (mg/kg).

FIG. 24B depicts the body weight changes and serum HMW adiponectinlevels in cynomolgus monkeys (N=3) that received a single i.v. injectionof BsAb17 at the indicated dose (mg/kg).

FIG. 24C depicts the EE of DIO mice (left: wt and right: adipoq KO) thatreceived single i.p. injection of indicated IgG (BsAb17) at 10 mg/kg(arrow). N=5˜6.

FIG. 24D depicts the various metabolic parameters in wt (+/+) and adipoqKO (−/−) DIO mice, which received single i.p. injection of indicated IgG(BsAb17) at 10 mg/kg. N=6. AUC: Area under the curve in GTT or ITT(T=0-2 h). p<0.1 (#), <0.05 (*), <0.005 (**) vs control.

FIG. 25 depicts the total RNA that was prepared from the mice describedin FIG. 19G using qPCR.

FIG. 26 depicts the level of ERK phosphorylation by BsAb17 in mousetissues. Tissues were harvested at 1 h after i.p. injection of leanC57BL/6 mice at 10 mg/kg BsAb17 or control IgG, and subjected toimmunohistochemistry using an antibody specific to phosphorylated ERK.Representative images from 2 animals are shown for each group. (1)Pancreas, (2) coronal brain section containing suprachiasmatic nuclei(arrow), (3) coronal brain section containing area postrema (triangularcollection of stained cells) and the central canal (arrow), and (4)coronal brain section containing median eminence (arrow). Note thatBsAb17-induced signal was apparent in the pancreatic acinar cells, butnot in any of the brain sections examined.

FIG. 27 depicts the normalization of HFD-induced hepatocyteproliferation by BsAb20. Hepatic BrdU incorporation in DIO mice treatedwith BsAb20 (1 or 3 mg/kg/week) or control IgG (3 mg/kg/week) for 8weeks or control lean C57BL/6 mice. * p<0.05 vs IgG-treated DIO mice(N=5˜8).

FIG. 28A is a schematic representation of the experiment shown in FIG.28B. DIO mice received BsAb20 (1 or 3 mg/kg/week) or control IgG (1mg/kg/week) for 6 weeks as indicated. Control lean C57BL/6 mice did notreceive treatment.

FIG. 28B depicts the bone phenotype after BsAb20 treatment. Femur andtibia were dissected and subjected to CT analysis. (N=7˜8). Note that nonegative effect was observed in various bone parameters in trabecularand cortical bones with the possible exception of cortical bonethickness, which showed a decreasing trend with 3 mg/kg/week BsAb20treatment although statistical significance was not reached. Since areduction in cortical bone thickness without an effect in trabecularbone density in calorie restricted mice has been reported (11), theobserved effect may be related to weight loss. p<0.001 (***), <0.01(**), <0.05 (*), <0.1 (#), <0.2 ($) vs DIO mice treated with controlIgG. N=7˜8.

FIG. 29 depicts the corticosterone levels in mice after BsAb17treatment. Serum corticosterone levels were measured at Zeitgeber time(ZT)=3 after euthanasia by decapitation. Control (CTRL) lean micereceived no treatment. Lipopolysaccharide (LPS) was i.p. injected intolean mice at 1 mg/kg at 3 hr prior to euthanasia (ZT=0) as a positivecontrol (12). IgG was i.p. injected into DIO mice at 5 or 25 mg/kg on 5days prior to euthanasia as indicated. Indicated statistical analysiswas conducted without LPS group. N=12.

FIG. 30 shows the binding of various different bispecific antibodieswith anti-FGFR1 and anti-KLB arms to cells expressing FGFR1c or FGFR1cand KLB.

FIG. 31 depicts binding of YW182.5 and YW182.5 derivatives to FGFR1proteins by ELISA.

DETAILED DESCRIPTION

For clarity and not by way of limitation the detailed description of thepresently disclosed subject matter is divided into the followingsubsections:

-   -   I. Definitions;    -   II. Antibodies;    -   III. Methods of Use;    -   IV. Pharmaceutical Formulations; and    -   V. Articles of Manufacture.

I. Definitions

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In certain embodiments, the number of amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less. In certain embodiments, the VL acceptorhuman framework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(d)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

“Klotho-beta,” “KLB” and “beta-Klotho,” as used herein, refers to anynative beta-Klotho from any vertebrate source, including mammals such asprimates (e.g., humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessed KLBas well as any form of KLB that results from processing in the cell. Theterm also encompasses naturally occurring variants of KLB, e.g., splicevariants or allelic variants. A non-limiting example of a human KLBamino acid sequence targeted by an antibody of the present disclosure,excluding the signal sequence, is as follows:

(SEQ ID NO: 145) FSGDGRAIWSKNPNFTPVNESQLFLYDTFPKNFFWGIGTGALQVEGSWKKDGKGPSIWDHFIHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYSTLLDALVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQMFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDIFKCQQSMVSVLGWFANPIHGDGDYPEGMRKKLFSVLPIFSEAEKHEMRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSRVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKESTPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKVISSRGFPFENSSSRCSQTQENTECTVCLFLVQKKPLIFLGCCFFSTLVLLLSIAIFQRQKRRKFWKAKNLQ HIPLKKGKRVVS.

In certain embodiments, a KLB protein can include a N-terminal signalsequence having the amino acid sequence

(SEQ ID NO: 157) MKPGCAAGSPGNEWIFFSTDEITTRYRNTMSNGGLQRSVILSALILLRA VTG.

The term “C-terminal domain of KLB” refers to the carboxy-terminalglycosidase-like domain of KLB. For example, the C-terminal domain ofthe exemplary KLB protein shown in SEQ ID NO: 145 comprises thefollowing amino acid sequence:

(SEQ ID NO: 155) FPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHADGWLNPSTAEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLGKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKV ISSRGFPFENSSSR.

The terms “anti-KLB antibody” and “an antibody that binds to KLB” referto an antibody that is capable of binding KLB with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting KLB. In one embodiment, the extent of binding of ananti-KLB antibody to an unrelated, non-KLB protein is less than about10% of the binding of the antibody to KLB as measured, e.g., by aradioimmunoassay (RIA). In certain embodiments, an antibody that bindsto KLB has a dissociation constant (K_(d)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g., from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M). In certainembodiments, an anti-KLB antibody binds to an epitope of KLB that isconserved among KLB from different species. In certain embodiments, ananti-KLB antibody binds to an epitope on KLB that is in the C-terminalpart of the protein.

The term “Fibroblast Growth Factor Receptor 1” or “FGFR1,” as usedherein, refers to any native FGFR1 from any vertebrate source, includingmammals such as primates (e.g., humans) and rodents (e.g., mice andrats), unless otherwise indicated. The term encompasses “full-length,”unprocessed FGFR1 as well as any form of FGFR1 that results fromprocessing in the cell. The term also encompasses naturally occurringvariants of FGFR1, e.g., splice variants or allelic variants, includingFGFR1c. A non-limiting example of a human FGFR1c amino acid is shownbelow:

(SEQ ID NO: 146) MWSWKCLLFWAVLVTATLCTARPSPTLPEQAQPWGAPVEVESFLVHPGDLLQLRCRLRDDVQSINWLRDGVQLAESNRTRITGEEVEVQDSVPADSGLYACVTSSPSGSDTTYFSVNVSDALPSSEDDDDDDDSSSEEKETDNTKPNPVAPYWTSPEKMEKKLHAVPAAKTVKFKCPSSGTPNPTLRWLKNGKEFKPDHRIGGYKVRYATWSIIMDSVVPSDKGNYTCIVENEYGSINHTYQLDVVERSPHRPILQAGLPANKTVALGSNVEFMCKVYSDPQPHIQWLKHIEVNGSKIGPDNLPYVQILKTAGVNTTDKEMEVLHLRNVSFEDAGEYTCLAGNSIGLSHHSAWLTVLEALEERPAVMTSPLYLEIIIYCTGAFLISCMVGSVIVYKMKSGTKKSDFHSQMAVHKLAKSIPLRRQVTVSADSSASMNSGVLLVRPSRLSSSGTPMLAGVSEYELPEDPRWELPRDRLVLGKPLGEGCFGQVVLAEAIGLDKDKPNRVTKVAVKMLKSDATEKDLSDLISEMEMMKMIGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLQARRPPGLEYCYNPSHNPEEQLSSKDLVSCAYQVARGMEYLASKKCIHRDLAARNVLVTEDNVMKIADFGLARDIHHIDYYKKTTNGRLPVKWMAPEALFDRIYTHQSDVWSFGVLLWEIFTLGGSPYPGVPVEELFKLLKEGHRMDKPSNCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRIVALTSNQEYLDLSMPLDQYSPSFPDTRSSTCSSGEDSVFSHEPLPEEPCLPRHPAQLANGGLKRR.

The terms “anti-FGFR1c antibody” refers to an antibody that is capableof binding FGFR1c with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting FGFR1c. Inone embodiment, the extent of binding of an anti-FGFR1c antibody to anunrelated, non-FGFR1c protein is less than about 10% of the binding ofthe antibody to FGFR1c as measured, e.g., by a radioimmunoassay (RIA).In certain embodiments, an antibody that binds to FGFR1c has adissociation constant (K_(d)) of ≤1 M, ≤100 mM, ≤10 mM, ≤1 mM, 100 M,≤10 M, ≤1 M, ≤100 nM, 10 nM, ≤1 nM, ≤0.1 nM, 0.01 nM, or ≤0.001 nM. Incertain embodiments, the K_(d) of an antibody that binds to FGFR1c,disclosed herein, can be 10⁻³ M or less, or 10⁻⁸ M or less, e.g., from10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M. In certain embodiments,an anti-FGFR1c antibody binds to an epitope of FGFR1c that is conservedamong FGFR1c from different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments.

An “antibody that competes for binding” with a reference antibody refersto an antibody that blocks binding of the reference antibody to itsantigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isdescribed in “Antibodies,” Harlow and Lane (Cold Spring Harbor Press,Cold Spring Harbor, NY).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.For example, and not by way of limitation, an “effective amount” canrefer to an amount of an antibody, disclosed herein, that is able toalleviate, minimize and/or prevent the symptoms of the disease and/ordisorder, prolong survival and/or prolong the period until relapse ofthe disease and/or disorder.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In certain embodiments, a human IgG heavy chain Fcregion extends from Cys226, or from Pro230, to the carboxyl-terminus ofthe heavy chain. However, the C-terminal lysine (Lys447) of the Fcregion may or may not be present. Unless otherwise specified herein,numbering of amino acid residues in the Fc region or constant region isaccording to the EU numbering system, also called the EU index, asdescribed in Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, M D, 1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” as usedinterchangeably herein, refer to cells into which exogenous nucleic acidhas been introduced, including the progeny of such cells. Host cellsinclude “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda MD (1991), Vols. 1-3. Incertain embodiments, for the VL, the subgroup is subgroup kappa I as inKabat et al., supra. In certain embodiments, for the VH, the subgroup issubgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence (“complementarity determining regions” or “CDRs”) and/or formstructurally defined loops (“hypervariable loops”) and/or contain theantigen-contacting residues (“antigen contacts”). Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.Generally, antibodies comprise six HVRs: three in the VH (H1, H2, H3),and three in the VL (L1, L2, L3). Exemplary HVRs herein include:

-   -   (a) hypervariable loops occurring at amino acid residues 26-32        (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101        (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));    -   (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56        (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3)        (Kabat et al., Sequences of Proteins of Immunological Interest,        5th Ed. Public Health Service, National Institutes of Health,        Bethesda, MD (1991));    -   (c) antigen contacts occurring at amino acid residues 27c-36        (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and        93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745        (1996)); and    -   (d) combinations of (a), (b), and/or (c), including HVR amino        acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),        26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102        (H3).

In certain embodiments, HVR residues comprise those identified in FIG.3A or FIG. 3B or elsewhere in the specification.

An “immunoconjugate” refers to an antibody conjugated to one or moreheterologous molecule(s), including but not limited to a cytotoxicagent.

An “individual” or “subject,” as used interchangeably herein, is amammal. Mammals include, but are not limited to, domesticated animals(e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans andnon-human primates such as monkeys), rabbits, and rodents (e.g., miceand rats). In certain embodiments, the individual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In certain embodiments, an antibody ispurified to greater than 95% or 99% purity as determined by, forexample, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF),capillary electrophoresis) or chromatographic (e.g., ion exchange orreverse phase HPLC). For review of methods for assessment of antibodypurity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an antibody” (including references to aspecific antibody, e.g., an anti-KLB antibody) refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody,” as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentlydisclosed subject matter may be made by a variety of techniques,including but not limited to the hybridoma method, recombinant DNAmethods, phage-display methods, and methods utilizing transgenic animalscontaining all or part of the human immunoglobulin loci, such methodsand other exemplary methods for making monoclonal antibodies beingdescribed herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert,” as used herein, refers to instructionscustomarily included in commercial packages of therapeutic products,that contain information about the indications, usage, dosage,administration, combination therapy, contraindications and/or warningsconcerning the use of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco,California, or may be compiled from the source code. The ALIGN-2 programshould be compiled for use on a UNIX operating system, including digitalUNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier,” as used herein, refers to aningredient in a pharmaceutical formulation, other than an activeingredient, which is nontoxic to a subject. A pharmaceuticallyacceptable carrier includes, but is not limited to, a buffer, excipient,stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of the individual being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In certain embodiments, antibodiesof the present disclosure can be used to delay development of a diseaseor to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

II. Antibodies

In one aspect, the invention is based, in part, on the discovery ofbispecific antibodies that bind to both KLB and FGFR1c and selectivelyactivate the FGFR1c/KLB receptor complex and induce the beneficialmetabolic changes expected from the FGF21-like activity, includingweight loss, and improvement in glucose and lipid metabolism, without asignificant impact on the liver and without loss in bone mass.

In certain embodiments, antibodies that bind to KLB are provided. Thepresent disclosure further provides anti-FGFR1 antibodies, e.g.,anti-FGFR1c antibodies. The present disclosure further providesbispecific antibodies that bind to both KLB and FGFR1 (referred toherein as anti-KLB/anti-FGFR1 bispecific antibodies). In certainembodiments, an anti-KLB/anti-FGFR1 bispecific antibody of the presentdisclosure binds to both KLB and FGFR1c. In certain embodiments, theantibodies of the present disclosure include antibodies that do notblock binding and/or interaction of the FGF ligands, e.g., FGF19 andFGF21, to the KLB/FGFR1c complex.

In certain embodiments, an antibody of the present disclosure does nothave a significant impact on the liver, e.g., liver function. Withoutbeing limited to a particular theory, an antibody of the presentdisclosure does not result in the activation of the FGFR1c/KLB receptorcomplex in the liver. In certain embodiments, an antibody of the presentdisclosure does not modulate the activity of an FGFR/KLB receptorcomplex in the liver as compared to the modulation of an FGFR/KLBreceptor complex in the liver by an FGF21 protein. In certainembodiments, an antibody of the present disclosure does not result inthe inhibition of the FGFR4/KLB complex and/or does not result in theelevation of liver enzymes such as, but not limited to, ALT, AST, ALPand GLDH. In certain embodiments, an antibody of the present disclosuredoes not function as an agonist of the FGFR2c/KLB complex and/or theFGFR3c/KLB complex in the liver, which can lead to activated MAPKsignaling and/or altered expression of Spry4 and Dusp6 in the liver.

In certain embodiments, an antibody of the present disclosure does notresult in the activation of MAPK signaling in the liver as compared tothe activation of MAPK signaling by an FGF21 protein. In certainembodiments, an antibody of the present disclosure does not function asan agonist of the FGFR4/KLB complex in the liver.

In certain embodiments, an antibody of the present disclosure can behumanized. In certain embodiments, an antibody of the present disclosurecomprises an acceptor human framework, e.g., a human immunoglobulinframework or a human consensus framework.

In certain embodiments, an antibody of the present disclosure can be amonoclonal antibody, including a chimeric, humanized or human antibody.In certain embodiments, an antibody of the present disclosure can be anantibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody, or F(ab′)₂fragment. In certain embodiments, the antibody is a full lengthantibody, e.g., an intact IgG1 antibody, or other antibody class orisotype as defined herein. In a certain embodiments, an antibody of thepresent disclosure can incorporate any of the features, singly or incombination, as described in Sections 1-7, detailed below.

Antibodies of the present disclosure are useful, e.g., for the diagnosisor treatment of metabolic disorders. Non-limiting examples of metabolicdisorders include polycystic ovary syndrome (PCOS), metabolic syndrome(MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholicfatty liver disease (NAFLD), hyperlipidemia, hypertension, type 2diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmunediabetes (LAD), maturity onset diabetes of the young (MODY), and agingand related diseases such as Alzheimer's disease, Parkinson's diseaseand ALS.

A. Exemplary Anti-KLB Antibodies

In one aspect, the present disclosure provides isolated antibodies thatbind to a KLB protein. In certain embodiments, an anti-KLB antibody ofthe present disclosure binds to the C-terminal domain of KLB. In certainembodiments, an anti-KLB antibody of the present disclosure binds to afragment of KLB that comprises the amino acid sequenceSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS (SEQ ID NO: 142). In certainembodiments, the antibody binds to the same epitope as an anti-KLBantibody, e.g., 8C5, described herein.

In certain embodiments, an anti-KLB antibody of the present disclosurecomprises at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising an amino acid sequence of any one of SEQ IDNOs: 1-15, e.g., 12 or 15; (b) HVR-H2 comprising an amino acid sequenceof any one of SEQ ID NOs: 16-31, e.g., 28 or 31; (c) HVR-H3 comprisingan amino acid sequence of any one of SEQ ID NOs: 32-47, e.g., 44 or 47;(d) HVR-L1 comprising an amino acid sequence of any one of SEQ ID NOs:48-62, e.g., 49 or 62; (e) HVR-L2 comprising an amino acid sequence ofany one of SEQ ID NOs: 63-78, e.g., 75 or 78; and (f) HVR-L3 comprisingan amino acid sequence of any one of SEQ ID NOs: 79-93, e.g., 90 or 93.

In certain embodiments, the present disclosure provides an anti-KLBantibody comprising at least one, two, three, four, five, or six HVRsselected from (a) HVR-H1 comprising SEQ ID NO: 12; (b) HVR-H2 comprisingSEQ ID NO: 28; (c) HVR-H3 comprising SEQ ID NO: 44; (d) HVR-L1comprising SEQ ID NO: 49; (e) HVR-L2 comprising SEQ ID NO: 75; and (f)HVR-L3 comprising SEQ ID NO: 90. In certain embodiments, the presentdisclosure provides an anti-KLB antibody comprising at least one, two,three, four, five, or six HVRs selected from (a) HVR-H1 comprising SEQID NO: 15; (b) HVR-H2 comprising SEQ ID NO 31; (c) HVR-H3 comprising SEQID NO: 47; (d) HVR-L1 comprising SEQ ID NO 62; (e) HVR-L2 comprising SEQID NO: 78; and (f) HVR-L3 comprising SEQ ID NO: 93.

The present disclosure further provides an anti-KLB antibody thatcomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 128. In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions as disclosed below), insertions, or deletionsrelative to the reference sequence, but an anti-KLB antibody comprisingthat sequence retains the ability to bind to KLB. In certainembodiments, a total of 1 to 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO: 128. In certain embodiments,substitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Alternatively or additionally, the anti-KLBantibody comprises the VH sequence in SEQ ID NO: 128, includingpost-translational modifications of that sequence as disclosed below. Incertain embodiments, the VH comprises one, two or three HVRs selectedfrom: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 15,(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 31, and (c)HVR-H3 comprising the amino acid sequence of SEQ ID NO: 47.

In another aspect, the present disclosure provides an anti-KLB antibody,wherein the antibody comprises a light chain variable domain (VL) havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO: 130. Incertain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-KLB antibody comprising that sequenceretains the ability to bind to KLB. In certain embodiments, a total of 1to 10 amino acids have been substituted, inserted and/or deleted in SEQID NO: 130. In certain embodiments, the substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Alternatively or additionally, the anti-KLB antibody comprises the VLsequence in SEQ ID NO: 130, including post-translational modificationsof that sequence. In certain embodiments, the VL comprises one, two orthree HVRs selected from (a) HVR-L1 comprising the amino acid sequenceof SEQ ID NO: 62; (b) HVR-L2 comprising the amino acid sequence of SEQID NO: 78; and (c) HVR-L3 comprising the amino acid sequence of SEQ IDNO: 93.

The present disclosure further provides an anti-KLB antibody, whereinthe antibody comprises a VH as in any of the embodiments provided above,and a VL as in any of the embodiments provided above. In certainembodiments, the antibody comprises the VH and VL sequences in SEQ IDNO: 128 and SEQ ID NO: 130, respectively, including post-translationalmodifications of those sequences.

In certain embodiments, an anti-KLB antibody binds to a fragment of KLBconsisting of the amino acid sequence SSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS(SEQ ID NO: 142).

B. Exemplary Anti-FGFR1 Antibodies

In one aspect, the present disclosure provides isolated antibodies thatbind to a FGFR1 protein. In certain embodiments, an anti-FGFR1 antibodyof the present disclosure binds to FGFR1c. In certain embodiments, thepresent disclosure provides an anti-FGFR1 antibody comprising at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising SEQ ID NO: 136; (b) HVR-H2 comprising SEQ ID NO: 137; (c)HVR-H3 comprising SEQ ID NO: 138; (d) HVR-L1 comprising SEQ ID NO: 139;(e) HVR-L2 comprising SEQ ID NO: 140; and (f) HVR-L3 comprising SEQ IDNO: 141.

In certain embodiments, an anti-FGFR1 antibody of the present disclosurecomprises a heavy chain variable domain (VH) sequence having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 132. In certainembodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-FGFR1 antibody comprising that sequenceretains the ability to bind to FGFR1. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 132. In certain embodiments, substitutions, insertions, ordeletions occur in regions outside the HVRs (i.e., in the FRs).Alternatively or additionally, the anti-FGFR1 antibody comprises the VHsequence in SEQ ID NO: 132, including post-translational modificationsof that sequence. In certain embodiments, the VH comprises one, two orthree HVRs selected from: (a) HVR-H1 comprising the amino acid sequenceof SEQ ID NO: 136, (b) HVR-H2 comprising the amino acid sequence of SEQID NO: 137, and (c) HVR-H3 comprising the amino acid sequence of SEQ IDNO: 138.

The present disclosure further provides an anti-FGFR1 antibody, whereinthe antibody comprises a light chain variable domain (VL) having atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO: 134. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-FGFR1 antibody comprising that sequenceretains the ability to bind to FGFR1. In certain embodiments, a total of1 to 10 amino acids have been substituted, inserted and/or deleted inSEQ ID NO: 134. In certain embodiments, the substitutions, insertions,or deletions occur in regions outside the HVRs (i.e., in the FRs).Alternatively or additionally, the anti-FGFR1 antibody comprises the VLsequence in SEQ ID NO: 134, including post-translational modificationsof that sequence. In a particular embodiment, the VL comprises one, twoor three HVRs selected from (a) HVR-L1 comprising the amino acidsequence of SEQ ID NO: 139; (b) HVR-L2 comprising the amino acidsequence of SEQ ID NO: 140; and (c) HVR-L3 comprising the amino acidsequence of SEQ ID NO: 141.

In another aspect, an anti-FGFR1 antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In certainembodiments, the anti-FGFR1 antibody comprises the VH and VL sequencesin SEQ ID NO: 132 and SEQ ID NO: 134, respectively, includingpost-translational modifications of those sequences.

In certain embodiments, an FGFR1 antibody of the present disclosurebinds to a fragment of FGFR1c consisting of the amino acid sequenceKLHAVPAAKTVKFKCP (SEQ ID NO: 143) or FKPDHRIGGYKVRY (SEQ ID NO: 144).

C. Exemplary Anti-KLB/Anti-FGFR1 Bispecific Antibodies

The present disclosure further provides bispecific antibodies that bindto both KLB and FGFR1 (i.e., anti-KLB/anti-FGFR1 bispecific antibodies).A bispecific antibody has two different binding specificities, see,e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243; Zeilder (1999) J. Immunol.163:1246-1252; Somasundaram (1999) Hum. Antibodies 9:47-54; Keler (1997)Cancer Res. 57:4008-4014. For example, and not by way of limitation, thepresently disclosed subject matter provides bispecific antibodies havingone binding site (e.g., antigen binding site) for a first epitopepresent on KLB and a second binding site for a second epitope present onFGFR1. For example, and not by way of limitation, the present disclosureprovides an antibody where one arm binds KLB and comprises any of theanti-KLB antibody sequences described herein and the second arm binds toFGFR1 and comprises any of the anti-FGFR1 antibody sequences describedherein. In certain embodiments, an anti-KLB/anti-FGFR1 bispecificantibody of the present disclosure has one binding site for a firstepitope present on KLB and a second binding site for a second epitopepresent on FGFR1c.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodydisclosed herein refers to an antibody that modulates KLB/FGFR1c complexactivity. For example, the bispecific anti-KLB/anti-FGFR1 bispecificantibody can function as an agonist and activate the KLB/FGFR1c complex.In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody is anantibody that increases the activity of the KLB/FGFR1c complex by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99% or 99.9%. Incertain embodiments, the anti-KLB/anti-FGFR1 bispecific can be anantibody that results in the phosphorylation of the downstream targetsof the KLB/FGFR1c complex, e.g., MAPK and/or ERK.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodydisclosed herein refers to an antibody that modulates KLB/FGFR1c complexactivity and does not block the interaction and/or binding of the nativeFGF ligands, e.g., FGF19 and FGF21, to the KLB/FGFR1c complex. Incertain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodydisclosed herein refers to an antibody that does not block the activityand/or binding of native FGF ligands to a FGF receptor in the absence ofKLB. For example, and not by way of limitation, an anti-KLB/anti-FGFR1bispecific antibody of the present disclosure does not block theinteraction of native FGF ligands with the FGFR1/KLA complex and/orFGFR1 alone. In certain embodiments, an anti-KLB/anti-FGFR1 bispecificantibody disclosed herein refers to an antibody that does not block theactivity and/or binding of native FGF ligands to KLB in the absence ofFGFR1. For example, and not by way of limitation, an anti-KLB/anti-FGFR1bispecific antibody of the present disclosure does not block theinteraction of native FGF ligands with the FGFR4/KLB complex, theFGFR2c/KLB complex and/or the FGFR3c/KLB complex.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody,e.g., an anti-KLB/anti-FGFR1c bispecific antibody, or an antigen-bindingportion thereof, includes a heavy chain and a light chain region. Incertain embodiments, the full length heavy chain includes amino acidshaving a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO:129. In certain embodiments, the full length light chain includes aminoacids having a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQID NO: 131. In certain embodiments, the full length heavy chain includesamino acids having the sequence set forth in SEQ ID NO: 129. In certainembodiments, the full length light chain includes amino acids having thesequence set forth in SEQ ID NO: 131.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody,e.g., an anti-KLB/anti-FGFR1c bispecific antibody, or an antigen-bindingportion thereof, includes a heavy chain variable region and a lightchain variable region. In certain embodiments, the heavy chain variableregion includes amino acids having a sequence that is at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence set forth in SEQ ID NO: 128. In certain embodiments, the lightchain variable region includes amino acids having a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 130. In certainembodiments, the heavy chain variable region includes amino acids havingthe sequence set forth in SEQ ID NO: 128. In certain embodiments, thelight chain variable region includes amino acids having the sequence setforth in SEQ ID NO: 130.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodycomprises at least one, two, three, four, five, or six HVRs selectedfrom (a) HVR-H1 comprising an amino acid sequence of any one of SEQ IDNOs: 1-15, e.g., 12 or 15; (b) HVR-H2 comprising an amino acid sequenceof any one of SEQ ID NOs: 16-31, e.g., 28 or 31; (c) HVR-H3 comprisingan amino acid sequence of any one of SEQ ID NOs: 32-47, e.g., 44 or 47;(d) HVR-L1 comprising an amino acid sequence of any one of SEQ ID NOs:48-62, e.g., 49 or 62; (e) HVR-L2 comprising an amino acid sequence ofany one of SEQ ID NOs: 63-78, e.g., 75 or 78; and (f) HVR-L3 comprisingan amino acid sequence of any one of SEQ ID NOs: 79-93, e.g., 90 or 93.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody,e.g., an anti-KLB/anti-FGFR1c bispecific antibody, comprises at leastone, two, three, four, five, or six HVRs selected from (a) HVR-H1comprising SEQ ID NO: 12; (b) HVR-H2 comprising SEQ ID NO: 28; (c)HVR-H3 comprising SEQ ID NO: 44; (d) HVR-L1 comprising SEQ ID NO: 49;(e) HVR-L2 comprising SEQ ID NO: 75; and (f) HVR-L3 comprising SEQ IDNO: 90. In certain embodiments, the present disclosure provides ananti-KLB antibody comprising at least one, two, three, four, five, orsix HVRs selected from (a) HVR-H1 comprising SEQ ID NO: 15; (b) HVR-H2comprising SEQ ID NO: 31; (c) HVR-H3 comprising SEQ ID NO: 47; (d)HVR-L1 comprising SEQ ID NO: 62; (e) HVR-L2 comprising SEQ ID NO: 78;and (f) HVR-L3 comprising SEQ ID NO: 93.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody,e.g., an anti-KLB/anti-FGFR1c bispecific antibody, includes a heavychain variable region that comprises CDR1, CDR2, and CDR3 domains, and alight chain variable region that comprises CDR1, CDR2, and CDR3 domains.In certain embodiments, the heavy chain variable region CDR1 domainincludes an amino acid sequence having a sequence set forth in SEQ IDNO: 1-15. In certain embodiments, the heavy chain variable region CDR2domain includes an amino acid sequence a sequence set forth in SEQ IDNO: 16-31. In certain embodiments, the heavy chain variable region CDR3domain includes an amino acid sequence having a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO: 32-47. In certain embodiments, the light chainvariable region CDR1 domain includes an amino acid sequence having asequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% identical to SEQ ID NO: 48-62. In certain embodiments, thelight chain variable region CDR2 domain includes an amino acid sequencehaving a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100% identical to SEQ ID NO: 63-78. In certainembodiments, the light chain variable region CDR3 domain includes anamino acid sequence having a sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:79-93.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody,e.g., an anti-KLB/anti-FGFR1c bispecific antibody, includes a heavychain variable region that comprises CDR1, CDR2, and CDR3 domains, and alight chain variable region that comprises CDR1, CDR2, and CDR3 domains.In certain embodiments, the heavy chain variable region CDR1 domainincludes an amino acid sequence having a sequence set forth in SEQ IDNO: 1-15. In certain embodiments, the heavy chain variable region CDR2domain includes an amino acid sequence having a sequence set forth inSEQ ID NO: 16-31. In certain embodiments, the heavy chain variableregion CDR3 domain includes an amino acid sequence having a sequence setforth in SEQ ID NO: 32-47. In certain embodiments, the light chainvariable region CDR1 domain includes an amino acid sequence having asequence set forth in SEQ ID NO: 48-62. In certain embodiments, thelight chain variable region CDR2 domain includes an amino acid sequencehaving a sequence set forth in SEQ ID NO: 63-78. In certain embodiments,the light chain variable region CDR3 domain includes an amino acidsequence having a sequence set forth in SEQ ID NO: 79-93.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody,e.g., an anti-KLB/anti-FGFR1c bispecific antibody, includes a heavychain variable region CDR1 having the sequence set forth in SEQ ID NO:15; a heavy chain variable region CDR2 having the sequence set forth inSEQ ID NO: 31; a heavy chain variable region CDR3 having the sequenceset forth in SEQ ID NO: 47; a light chain variable region CDR1 havingthe sequence set forth in SEQ ID NO: 62; a light chain variable regionCDR2 having the sequence set forth in SEQ ID NO: 78; and a light chainvariable region CDR3 having the sequence set forth in SEQ ID NO: 93.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodyincludes a first antibody, or antigen binding portion thereof, andincludes a second antibody, or antigen binding portion thereof, wherethe first antibody, or antigen binding portion thereof, binds to anepitope present on KLB, and the second antibody, or antigen bindingportion thereof, bind to an epitope present on FGFR1, e.g., FGFR1c. Forexample, and not by way of limitation, the first antibody, or antigenbinding portion thereof, can include a heavy chain variable region and alight chain variable region; and the second antibody, or antigen bindingportion thereof, can include a heavy chain variable region and a lightchain variable region. In certain embodiments, the heavy chain variableregion of the first antibody, or antigen binding portion thereof,includes amino acids having a sequence that is at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence setforth in SEQ ID NO: 128. In certain embodiments, the light chainvariable region of the first antibody, or antigen binding portionsthereof, includes amino acids having a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence set forth in SEQ ID NO: 130. In certain embodiments, the heavychain variable region of the second antibody or antigen binding portionthereof includes amino acids having a sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence set forth in SEQ ID NO: 132. In certain embodiments, the lightchain variable region of the second antibody, or antigen bindingportions thereof, includes amino acids having a sequence that is atleast 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the sequence set forth in SEQ ID NO: 134.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody thatbinds to the same epitope as an anti-KLB antibody is provided herein.For example, in certain embodiments, an anti-KLB/anti-FGFR1 bispecificantibody is provided that binds to the same epitope as an anti-KLBantibody comprising the VH sequence of SEQ ID NO: 128 and a VL sequenceof SEQ ID NO: 130. In certain embodiments, an anti-KLB/anti-FGFR1bispecific antibody is provided that binds to a fragment of KLBconsisting of the amino acid sequence

(SEQ ID NO: 142) SSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody isprovided that binds to a fragment of KLB having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the sequence set forth in SEQ ID NO: 142.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody bindsto the same epitope as an anti-KLB antibody is provided herein. Forexample, in certain embodiments, an anti-KLB/anti-FGFR1 bispecificantibody is provided that binds to the same epitope as an anti-KLBantibody comprising the full length heavy chain sequence of SEQ ID NO:129 and a full length light chain sequence of SEQ ID NO: 131.

In certain embodiments, the present disclosure provides ananti-KLB/anti-FGFR1 bispecific antibody that binds to the same epitopeas an anti-FGFR1 antibody provided herein. For example, in certainembodiments, an anti-KLB/anti-FGFR1 bispecific antibody is provided thatbinds to the same epitope as an anti-FGFR1 antibody comprising the VHsequence of SEQ ID NO: 132 and a VL sequence of SEQ ID NO: 134. Incertain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody isprovided that binds to a fragment of FGFR1c comprising amino acidsequence KLHAVPAAKTVKFKCP (SEQ ID NO: 143) or FKPDHRIGGYKVRY (SEQ ID NO:144).

In certain embodiments, the present disclosure provides ananti-KLB/anti-FGFR1 bispecific antibody that binds to the same epitopeas an anti-FGFR1 antibody provided herein. For example, in certainembodiments, an anti-KLB/anti-FGFR1 bispecific antibody is provided thatbinds to the same epitope as an anti-FGFR1 antibody comprising the heavychain sequence of SEQ ID NO: 133 and a light chain sequence of SEQ IDNO: 135.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody ofthe present disclosure binds to a fragment of FGFR1c having an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 143.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody ofthe present disclosure binds to a fragment of FGFR1c having an aminoacid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 144.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody isprovided that binds to a fragment of KLB having an amino acid sequencethat is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the sequence set forth in SEQ ID NO: 142, and binds toa fragment of FGFR1c having an amino acid sequence that is at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to thesequence set forth in SEQ ID NO: 143 or 144.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody isprovided that binds to a fragment of KLB having the amino acid sequenceset forth in SEQ ID NO: 142 and binds to a fragment of FGFR1c having theamino acid sequence set forth in SEQ ID NO: 143 or 144.

1. Antibody Affinity

In certain embodiments, an antibody of the present disclosure can have adissociation constant (K_(d)) of ≤1 M, ≤100 mM, ≤10 mM, ≤1 mM, ≤100 μM,≤10 M, ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM.In certain embodiments, an antibody of the present disclosure can have aK_(d) of about 10³ or less, or 10⁻⁸M or less, e.g., from 10⁻⁸M to 10-13M, e.g., from 10⁻⁹ M to 10-13 M.

In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibody caninclude an anti-FGFR1 arm that has a K_(d) of about 10 nM to about 10μM. In certain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodywith an FGFR1 arm that has a low affinity can mitigate the risk of theanti-KLB/anti-FGFR1 bispecific antibody from binding to FGFR1 tightly inthe absence of KLB and preventing the binding and/or activation of FGFR1by other FGF ligands such as, but not limited to, FGF1, FGF2, FGF8 andFGF23. In certain embodiments, an FGFR1 arm with a low affinity canpermit the presence of higher levels of anti-FGFR1 impurities such as,but not limited to, anti-FGFR1 half-knob antibodies, non-covalentanti-FGFR1 dimers, covalent anti-FGFR1 dimers and high-molecular weightspecies, without resulting in clinically significant side effects. Forexample, in certain embodiments, approximately 2% high molecular weightspecies and 1.5% anti-FGFR1 half-antibody can be present in apreparation of an anti-KLB/anti-FGFR1 bispecific antibody of the presentdisclosure without resulting in adverse biological effects.

In certain embodiments, K_(d) can be measured by a radiolabeled antigenbinding assay (RIA). In certain embodiments, an RIA can be performedwith a Fab version of an antibody of interest and its antigen. Forexample, and not by way of limitation, a solution binding affinity ofFabs for antigen is measured by equilibrating Fab with a minimalconcentration of (¹²⁵I)-labeled antigen in the presence of a titrationseries of unlabeled antigen, then capturing bound antigen with ananti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

In certain embodiments, K_(d) can be measured using a BIACORE® surfaceplasmon resonance assay. For example, and not by way of limitation, anassay using a BIACORE®-2000 or a BIACORE®-3000 (Biacore, Inc.,Piscataway, NJ) is performed at 25° C. with immobilized antigen CM5chips at ˜10 response units (RU). In certain embodiments,carboxymethylated dextran biosensor chips (CM5, Biacore, Inc.) areactivated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, to 5 μg/ml (˜0.2 μM) before injection at a flow rate of 5μl/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1 M ethanolamine isinjected to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flowrate of approximately 25 μl/min. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(d)) can be calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 106 M⁻¹ s⁻¹ by the surface plasmonresonance assay above, then the on-rate can be determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™spectrophotometer (ThermoSpectronic) with a stirred cuvette.

2. Antibody Fragments

In certain embodiments, an antibody of the present disclosure is anantibody fragment. Antibody fragments include, but are not limited to,Fab, Fab′, Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragmentsdescribed below. For a review of certain antibody fragments, see Hudsonet al. Nat. Med 9:129-134 (2003). For a review of scFv fragments, see,e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

In certain embodiments, an antibody of the present disclosure can be adiabody. Diabodies are antibody fragments with two antigen-binding sitesthat may be bivalent or bispecific. See, for example, EP 404,097; WO1993/01161; Hudson et al., Nat. Med 9:129-134 (2003); and Hollinger etal., Proc. Natl. Acad Sci. USA 90: 6444-6448 (1993). Triabodies andtetrabodies are also described in Hudson et al., Nat. Med 9:129-134(2003).

In certain embodiments, an antibody of the present disclosure can be asingle-domain antibody. Single-domain antibodies are antibody fragmentsthat comprise all or a portion of the heavy chain variable domain or allor a portion of the light chain variable domain of an antibody. Incertain embodiments, a single-domain antibody is a human single-domainantibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No.6,248,516 B1).

Antibody fragments can be made by various techniques including, but notlimited to, proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g., E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody of the present disclosure is achimeric antibody. Certain chimeric antibodies are described, e.g., inU.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci.USA, 81:6851-6855 (1984)). In certain embodiments, a chimeric antibodyof the present disclosure comprises a non-human variable region (e.g., avariable region derived from a mouse, rat, hamster, rabbit, or non-humanprimate, such as a monkey) and a human constant region. In a furtherexample, a chimeric antibody can be a “class switched” antibody in whichthe class or subclass has been changed from that of the parent antibody.Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, a chimeric antibody of the present disclosurecan be a humanized antibody. Typically, a non-human antibody ishumanized to reduce immunogenicity to humans, while retaining thespecificity and affinity of the parental non-human antibody. Generally,a humanized antibody comprises one or more variable domains in whichHVRs, e.g., CDRs, (or portions thereof) are derived from a non-humanantibody, and FRs (or portions thereof) are derived from human antibodysequences. A humanized antibody optionally will also comprise at least aportion of a human constant region. In certain embodiments, some FRresidues in a humanized antibody are substituted with correspondingresidues from a non-human antibody (e.g., the antibody from which theHVR residues are derived), e.g., to restore or improve antibodyspecificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing specificity determining region(SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing“resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing“FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimkaet al., Br. J. Cancer, 83:252-260 (2000) (describing the “guidedselection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody of the present disclosure can be ahuman antibody. Human antibodies can be produced using varioustechniques known in the art. Human antibodies are described generally invan Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) andLonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies can be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the present disclosure can be isolated by screeningcombinatorial libraries for antibodies with the desired activity oractivities. For example, a variety of methods are known in the art forgenerating phage display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Such methodsare reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N J, 2001) andfurther described, e.g., in the McCafferty et al., Nature 348:552-554;Clackson et al., Nature 352: 624-628 (1991); Marks et al., J Mol. Biol.222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology248:161-175 (Lo, ed., Human Press, Totowa, N J, 2003); Sidhu et al., JMol. Biol. 338(2): 299-310 (2004); Lee et al., J Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). In certain embodiments, naive libraries can also be madesynthetically by cloning unrearranged V-gene segments from stem cells,and using PCR primers containing random sequence to encode the highlyvariable CDR3 regions and to accomplish rearrangement in vitro, asdescribed by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).Patent publications describing human antibody phage libraries include,for example: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody of the present disclosure can be amultispecific antibody, e.g., a bispecific antibody. Multispecificantibodies are monoclonal antibodies that have binding specificities forat least two different epitopes. In certain embodiments, one of thebinding specificities is for an epitope present on KLB and the other isfor any other antigen. In certain embodiments, one of the bindingspecificities is for an epitope present on FGFR1 and the other is forany other antigen. In certain embodiments, a bispecific antibody of thepresent disclosure can bind an epitope on KLB and can bind an epitope onFGFR1. In certain embodiments, a bispecific antibody of the presentdisclosure can bind an epitope on KLB and can bind an epitope on FGFR1c.Bispecific antibodies can be prepared as full length antibodies orantibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g., US 2006/0025576A1).

7. Antibody Variants

The presently disclosed subject matter further provides amino acidsequence variants of the disclosed antibodies. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of an antibodycan be prepared by introducing appropriate modifications into thenucleotide sequence encoding the antibody, or by peptide synthesis. Suchmodifications include, but are not limited to, deletions from, and/orinsertions into and/or substitutions of residues within the amino acidsequences of the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final antibody, i.e., modified, possesses the desiredcharacteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants can have one or more aminoacid substitutions. Sites of interest for substitutional mutagenesisinclude the HVRs and FRs. Non-limiting examples of conservativesubstitutions are shown in Table 1 under the heading of “preferredsubstitutions.” Non-limiting examples of more substantial changes areprovided in Table 1 under the heading of “exemplary substitutions,” andas further described below in reference to amino acid side chainclasses. Amino acid substitutions can be introduced into an antibody ofinterest and the products screened for a desired activity, e.g.,retained/improved antigen binding, decreased immunogenicity or improvedcomplement dependent cytotoxicity (CDC) or antibody-dependentcell-mediated cytotoxicity (ADCC).

TABLE 1 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine LeuAmino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

In certain embodiments, non-conservative substitutions will entailexchanging a member of one of these classes for another class.

In certain embodiments, a type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody, e.g., a humanized or human antibody. Generally, the resultingvariant(s) selected for further study will have modifications, e.g.,improvements, in certain biological properties such as, but not limitedto, increased affinity, reduced immunogenicity, relative to the parentantibody and/or will have substantially retained certain biologicalproperties of the parent antibody. A non-limiting example of asubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g., bindingaffinity).

In certain embodiments, alterations (e.g., substitutions) can be made inHVRs, e.g., to improve antibody affinity. Such alterations may be madein HVR “hotspots,” i.e., residues encoded by codons that undergomutation at high frequency during the somatic maturation process (see,e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residuesthat contact antigen, with the resulting variant VH or VL being testedfor binding affinity. Affinity maturation by constructing andreselecting from secondary libraries has been described, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, NJ, (2001)). In certain embodiments ofaffinity maturation, diversity can be introduced into the variable geneschosen for maturation by any of a variety of methods (e.g., error-pronePCR, chain shuffling, or oligonucleotide-directed mutagenesis). Asecondary library is then created. The library is then screened toidentify any antibody variants with the desired affinity. Another methodto introduce diversity involves HVR-directed approaches, in whichseveral HVR residues (e.g., 4-6 residues at a time) are randomized. HVRresidues involved in antigen binding can be specifically identified,e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions canoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may, for example, be outside ofantigen contacting residues in the HVRs. In certain embodiments of thevariant VH and VL sequences provided above, each HVR either isunaltered, or contains no more than one, two or three amino acidsubstitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for Antibody-directedenzyme prodrug therapy (ADEPT)) or a polypeptide which increases theserum half-life of the antibody.

b) Glycosylation Variants

In certain embodiments, an antibody of the present disclosure can bealtered to increase or decrease the extent to which the antibody isglycosylated. Addition or deletion of glycosylation sites to an antibodymay be conveniently accomplished by altering the amino acid sequencesuch that one or more glycosylation sites is created or removed.

In certain embodiments, where the antibody comprises an Fc region, thecarbohydrate attached thereto can be altered. Native antibodies producedby mammalian cells typically comprise a branched, biantennaryoligosaccharide that is generally attached by an N-linkage to Asn297 ofthe CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH15:26-32 (1997). The oligosaccharide may include various carbohydrates,e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialicacid, as well as a fucose attached to a GlcNAc in the “stem” of thebiantennary oligosaccharide structure. In certain embodiments,modifications of the oligosaccharide in an antibody of the presentdisclosure can be made in order to create antibody variants with certainimproved properties.

In certain embodiments, antibody variants are provided having acarbohydrate structure that lacks fucose attached (directly orindirectly) to an Fc region. For example, the amount of fucose in suchantibody can be from about 1% to about 80%, from about 1% to about 65%,from about 5% to about 65% or from about 20% to about 40% and values inbetween.

In certain embodiments, the amount of fucose can be determined bycalculating the average amount of fucose within the sugar chain atAsn297, relative to the sum of all glycostructures attached to Asn 297(e.g., complex, hybrid and high mannose structures) as measured byMALDI-TOF mass spectrometry, as described in WO 2008/077546, forexample. Asn297 refers to the asparagine residue located at aboutposition 297 in the Fc region (Eu numbering of Fc region residues);however, Asn297 can also be located about +3 amino acids upstream ordownstream of position 297, i.e., between positions 294 and 300, due tominor sequence variations in antibodies. Such fucosylation variants mayhave improved ADCC function. See, e.g., US Patent Publication Nos. US2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: US 2003/0157108; WO2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004).

Defucosylated antibodies can be produced in any cell line that aredeficient in protein fucosylation. Non-limiting examples of cell linesinclude Lec13 CHO cells deficient in protein fucosylation (Ripka et al.Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibodies variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Non-limitingexamples of such antibody variants are described, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana etal.); and US 2005/0123546 (Umana et al.). Antibody variants with atleast one galactose residue in the oligosaccharide attached to the Fcregion are also provided. Such antibody variants can have improved CDCfunction. Such antibody variants are described, e.g., in WO 1997/30087(Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications can beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g., a substitution) atone or more amino acid positions.

In certain embodiments, the present disclosure provides an antibodyvariant that possesses some but not all effector functions, which makeit a desirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.,Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods can be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (Cell Technology, Inc. Mountain View, CA; and CYTOTOX 96®non-radioactive cytotoxicity assay (Promega, Madison, WI). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays canalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay can be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)). In certain embodiments, alterations can be made in the Fcregion that result in altered (i.e., either improved or diminished) C1qbinding and/or Complement Dependent Cytotoxicity (CDC), e.g., asdescribed in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al.J. Immunol. 164: 4178-4184 (2000).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).

In certain embodiments, an antibody variant of the present disclosurecomprises an Fc region with one or more amino acid substitutions whichimprove ADCC, e.g., substitutions at positions 298, 333, and/or 334 ofthe Fc region (EU numbering of residues).

In certain embodiments, alteration made in the Fc region of an antibody,e.g., a bispecific antibody, disclosed herein, can produce a variantantibody with an increased half-life and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein, which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies can be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody of the present disclosure can befurther modified to contain additional nonproteinaceous moieties thatare known in the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In certain embodiments, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). In certain embodiments, the radiation can be of any wavelength,and includes, but is not limited to, wavelengths that do not harmordinary cells, but which heat the nonproteinaceous moiety to atemperature at which cells proximal to the antibody-nonproteinaceousmoiety are killed.

D. Methods of Antibody Production

The antibodies disclosed herein can be produced using any available orknown technique in the art. For example, but not by way of limitation,antibodies can be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. Detailed procedures togenerate antibodies are described in the Examples below.

The presently disclosed subject matter further provides an isolatednucleic acid encoding an antibody disclosed herein. For example, theisolated nucleic acid can encode an amino acid sequence that includesthe VL and/or an amino acid sequence comprising the VH of the antibody,e.g., the light and/or heavy chains of the antibody. In certainembodiments, the isolated nucleic acid can include a nucleotide sequencethat encodes a heavy chain variable region amino acid sequence havingthe sequence set forth in SEQ ID NO: 128, and/or a nucleotide sequencethat encodes a light chain variable region amino acid sequence havingthe sequence set forth in SEQ ID NO: 130.

In certain embodiments, the nucleic acid can be present in one or morevectors, e.g., expression vectors. As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. Another type of vector isa viral vector, where additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors,expression vectors, are capable of directing the expression of genes towhich they are operably linked. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids(vectors). However, the disclosed subject matter is intended to includesuch other forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses) that serve equivalent functions.

In certain embodiments, the nucleic acid encoding an antibody of thepresent disclosure and/or the one or more vectors including the nucleicacid can be introduced into a host cell. In certain embodiments, theintroduction of a nucleic acid into a cell can be carried out by anymethod known in the art including, but not limited to, transfection,electroporation, microinjection, infection with a viral or bacteriophagevector containing the nucleic acid sequences, cell fusion,chromosome-mediated gene transfer, microcell-mediated gene transfer,spheroplast fusion, etc. In certain embodiments, a host cell caninclude, e.g., has been transformed with: (1) a vector comprising anucleic acid that encodes an amino acid sequence comprising the VL ofthe antibody and an amino acid sequence comprising the VH of theantibody, or (2) a first vector comprising a nucleic acid that encodesan amino acid sequence comprising the VL of the antibody and a secondvector comprising a nucleic acid that encodes an amino acid sequencecomprising the VH of the antibody. In certain embodiments, the host cellis eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell(e.g., Y0, NS0, Sp20 cell).

In certain embodiments, the methods of making an anti-KLB antibody oranti-FGFR1c can include culturing a host cell, in which a nucleic acidencoding the antibody has been introduced, under conditions suitable forexpression of the antibody, and optionally recovering the antibody fromthe host cell and/or host cell culture medium. In certain embodiments,the antibody is recovered from the host cell through chromatographytechniques.

For recombinant production of an antibody of the present disclosure, anucleic acid encoding an antibody, e.g., as described above, can beisolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Such nucleic acid may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies can be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006). Suitable host cells for theexpression of glycosylated antibody can also derived from multicellularorganisms (invertebrates and vertebrates). Examples of invertebratecells include plant and insect cells. Numerous baculoviral strains havebeen identified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

In certain embodiments, plant cell cultures can be utilized as hostcells. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants).

In certain embodiments, vertebrate cells can also be used as hosts. Forexample, and not by way of limitation, mammalian cell lines that areadapted to grow in suspension can be useful. Non-limiting examples ofuseful mammalian host cell lines are monkey kidney CV1 line transformedby SV40 (COS-7); human embryonic kidney line (293 or 293 cells asdescribed, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described,e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells(CV1); African green monkey kidney cells (VERO-76); human cervicalcarcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat livercells (BRL 3A); human lung cells (W138); human liver cells (Hep G2);mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,NJ), pp. 255-268 (2003).

In certain embodiments, techniques for making bispecific and/ormultispecific antibodies include, but are not limited to, recombinantco-expression of two immunoglobulin heavy chain-light chain pairs havingdifferent specificities (see Milstein and Cuello, Nature 305: 537(1983)), PCT Patent Application No. WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Bispecific antibodies can also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebispecific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Bispecific and multispecific molecules of the present disclosure canalso be made using chemical techniques (see, e.g., Kranz (1981) Proc.Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (see, e.g., U.S.Pat. No. 4,474,893), or recombinant DNA techniques. Bispecific andmultispecific molecules of the presently disclosed subject matter canalso be prepared by conjugating the constituent binding specificities,e.g., a first epitope and a second epitope binding specificities, usingmethods known in the art and as described herein. For example, and notby way of limitation, each binding specificity of the bispecific andmultispecific molecule can be generated separately and then conjugatedto one another. When the binding specificities are proteins or peptides,a variety of coupling or cross-linking agents can be used for covalentconjugation. Non-limiting examples of cross-linking agents includeprotein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1-carboxylate(sulfo-SMCC) (see, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu(1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include thosedescribed by Paulus (BehringIns. Mitt. (1985) No. 78, 118-132; Brennan(1985) Science 229:81-83), Glennie (1987) J. Immunol. 139: 2367-2375).When the binding specificities are antibodies (e.g., two humanizedantibodies), they can be conjugated via sulfhydryl bonding of theC-terminus hinge regions of the two heavy chains. In certainembodiments, the hinge region can be modified to contain an odd numberof sulfhydryl residues, e.g., one, prior to conjugation.

In certain embodiments, both binding specificities of a bispecificantibody can be encoded in the same vector and expressed and assembledin the same host cell. This method is particularly useful where thebispecific and multispecific molecule is a MAb×MAb, MAb×Fab, Fab×F(ab′)₂or ligand×Fab fusion protein. In certain embodiments, a bispecificantibody of the present disclosure can be a single chain molecule, suchas a single chain bispecific antibody, a single chain bispecificmolecule comprising one single chain antibody and a binding determinant,or a single chain bispecific molecule comprising two bindingdeterminants. Bispecific and multispecific molecules can also be singlechain molecules or can comprise at least two single chain molecules.Methods for preparing bi- and multispecific molecules are described, forexample, in U.S. Pat. Nos. 5,260,203; 5,455,030; 4,881,175; 5,132,405;5,091,513; 5,476,786; 5,013,653; 5,258,498; and 5,482,858. Engineeredantibodies with three or more functional antigen binding sites (e.g.,epitope binding sites) including “Octopus antibodies,” are also includedherein (see, e.g., US 2006/0025576A1).

The present disclosure further provides tri-specific, e.g.,tri-functional, antibodies. For example, and not by way of limitation, atri-specific antibody of the present disclosure can bind to and/orinteract with an epitope present on KLB, an epitope present on FGFR1,and an epitope or antigen present on a third protein such as, but notlimited to, PCSK9, GCGR, AdipoR, ZnT8, ApoL1, MSTN, InsR or FABP4.

In certain embodiments, an animal system can be used to produce anantibody of the present disclosure. One animal system for preparinghybridomas is the murine system. Hybridoma production in the mouse is avery well established procedure. Immunization protocols and techniquesfor isolation of immunized splenocytes for fusion are known in the art.Fusion partners (e.g., murine myeloma cells) and fusion procedures arealso known (see, e.g., Harlow and Lane (1988), Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NewYork).

E. Assays

The antibodies of the present disclosure provided herein can beidentified, screened for, or characterized for their physical/chemicalproperties and/or biological activities by various assays known in theart and provided herein.

1. Binding Assays and Other Assays

In certain embodiments, an antibody of the present disclosure can testedfor its antigen binding activity by known methods, such enzyme-linkedimmunosorbent assay (ELISA), a radioimmunoassay (RIA), or a Western BlotAssay. Each of these assays generally detects the presence ofprotein-antibody complexes of particular interest by employing a labeledreagent (e.g., an antibody) specific for the complex of interest. Forexample, the KLB-antibody complexes can be detected using, e.g., anenzyme-linked antibody or antibody fragment which recognizes andspecifically binds to the antibody-KLB complexes. Alternatively, thecomplexes can be detected using any of a variety of other immunoassays.For example, the antibody can be radioactively labeled and used in aradioimmunoassay (RIA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by such meansas the use of a Geiger counter or a scintillation counter or byautoradiography.

In certain embodiments, competition assays can be used to identify anantibody that competes with an anti-KLB antibody of the presentdisclosure, e.g., 12A11 or 8C5, for binding to KLB. In certainembodiments, such a competing antibody binds to the same epitope (e.g.,a linear or a conformational epitope) that is bound by 12A11 or 8C5.Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

In a non-limiting example of a competition assay, immobilized KLB can beincubated in a solution comprising a first labeled antibody that bindsto KLB (e.g., 12A11 or 8C5) and a second unlabeled antibody that isbeing tested for its ability to compete with the first antibody forbinding to KLB. The second antibody may be present in a hybridomasupernatant. As a control, immobilized KLB is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to KLB, excess unbound antibody is removed, and theamount of label associated with immobilized KLB is measured. If theamount of label associated with immobilized KLB is substantially reducedin the test sample relative to the control sample, then that indicatesthat the second antibody is competing with the first antibody forbinding to KLB. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

2. Activity Assays

The present disclosure provides assays for identifying anti-KLBantibodies thereof having biological activity. Biological activity mayinclude, e.g., activating a KLB/FGFR1c receptor complex. Antibodieshaving such biological activity in vivo and/or in vitro are alsoprovided. In certain embodiments, the assays can include bindingantibodies of the present disclosure to cells, e.g., 293T cellsexpressing KLB, and analyzing the activity and/or phosphorylation statesof one or more downstream targets of the KLB-FGFR1c receptor complex,e.g., ERK. In certain embodiments, the assay can include theadministering of an antibody of the present disclosure to a subject,e.g., a non-human animal, and analyzing the effect the antibody has onthe glucose level in the subject.

F. Immunoconjugates

The presently disclosed subject matter further provides immunoconjugatescomprising an antibody, disclosed herein, conjugated to one or morecytotoxic agents, such as chemotherapeutic agents or drugs, growthinhibitory agents, toxins (e.g., protein toxins, enzymatically activetoxins of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or radioactive isotopes. For example, an antibody orantigen-binding portion of the disclosed subject matter can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic.

In certain embodiments, an immunoconjugate is an antibody-drug conjugate(ADC) in which an antibody is conjugated to one or more drugs, includingbut not limited to a maytansinoid (see U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235); an auristatin such asmonomethylauristatin drug moieties DE and DF (MMAE and MMAF) (see U.S.Pat. Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; acalicheamicin or derivative thereof (see U.S. Pat. Nos. 5,712,374,5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode etal., Cancer Res. 58:2925-2928 (1998)); an anthracycline such asdaunomycin or doxorubicin (see Kratz et al., Current Med Chem.13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagyet al., Proc. Natl. Acad Sci. USA 97:829-834 (2000); Dubowchik et al.,Bioorg. & Med Chem. Letters 12:1529-1532 (2002); King et al., J. MedChem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate;vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel,and ortataxel; a trichothecene; and CC1065.

In certain embodiments, an immunoconjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), Momordica charantiainhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In certain embodiments, an immunoconjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Non-limiting examples include At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu. When the radioconjugate is used for detection, it caninclude a radioactive atom for scintigraphic studies, for example tc99mor 1123, or a spin label for nuclear magnetic resonance (NMR) imaging(also known as magnetic resonance imaging, mri), such as iodine-123again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,oxygen-17, gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent can be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker can be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) can be used.

The immunuoconjugates or ADCs herein expressly contemplate, but are notlimited to, such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

II. Methods of Use

The presently disclosed subject matter further provides methods forusing the disclosed antibodies, e.g., an anti-KLB/anti-FGFR1c bispecificantibody. In certain embodiments, the methods are directed totherapeutic uses of the presently disclosed antibodies. In certainembodiments, the methods are directed to the use of the disclosedantibodies in diagnostic methods.

A. Diagnostic and Detection Methods

In certain embodiments, any antibody disclosed herein that hasspecificity for KLB, e.g., an anti-KLB antibody and/or ananti-KLB/anti-FGFR1 bispecific antibody, disclosed above, can be usefulfor detecting the presence of KLB in a biological sample. In a furtheraspect, the presently disclosed subject matter provides methods fordiagnosing and/or detecting a disease using an anti-KLB antibody or ananti-KLB/anti-FGFR1 bispecific antibody, disclosed herein. The term“detecting,” as used herein, encompasses quantitative and/or qualitativedetection.

In certain non-limiting embodiments, a biological sample includes, butis not limited to, a clinical sample, one or more cells, cells inculture, cell supernatants, cell lysates and tissue samples. The sourceof the sample may be solid tissue (e.g., from a fresh, frozen, and/orpreserved organ, tissue sample, biopsy, or aspirate) or cells from theindividual. In certain embodiments, a biological sample can include oneor more cells and/or tissue from a liver, e.g., from a liver of asubject.

In certain embodiments, an anti-KLB antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of KLB in a biological sample is provided. Incertain embodiments, the method of diagnosis or detection includescontacting a biological sample with an antibody that binds an epitopepresent on KLB, as described herein, under conditions permissive forbinding of the antibody to KLB, and detecting whether a complex isformed between the antibody and KLB. Such method may be an in vitro orin vivo method, e.g., immunofluorescence or western blot. In certainembodiments, an anti-KLB antibody is used to select subjects eligiblefor therapy with an anti-KLB antibody, e.g., where KLB is a biomarkerfor selection of patients.

In certain embodiments, an antibody of the present disclosure, e.g., ananti-KLB/anti-FGFR1 bispecific antibody, for use in the disclosedmethods does not have a significant impact on the liver, e.g., liverfunction. In certain embodiments, an antibody of the present disclosuredoes not modulate the activity of an FGFR/KLB receptor complex in theliver as compared to the modulation of an FGFR/KLB receptor complex inthe liver by an FGF21 protein. In certain embodiments, an antibody ofthe present disclosure does not result in the inhibition of theFGFR4/KLB complex and/or does not result in the elevation of liverenzymes such as, but not limited to, ALT, AST, ALP and GLDH. In certainembodiments, an antibody of the present disclosure does not function asan agonist of the FGFR2c/KLB complex and/or the FGFR3c/KLB complex inthe liver, which can lead to activated MAPK signaling and/or alteredexpression of Spry4 and Dusp6 in the liver. In certain embodiments, anantibody of the present disclosure does not result in the activation ofMAPK signaling in the liver as compared to the activation of MAPKsignaling by an FGF21 protein. In certain embodiments, an antibody ofthe present disclosure does not function as an agonist of the FGFR4/KLBcomplex in the liver.

In certain embodiments, an antibody of the present disclosure, e.g., ananti-KLB/anti-FGFR1 bispecific antibody, for use in the disclosedmethods include antibodies that do not block binding and/or interactionof the FGF ligands, e.g., FGF19 and FGF21, to the KLB/FGFR1c complex. Incertain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodydisclosed herein refers to an antibody that modulates KLB/FGFR1c complexactivity and does not block the interaction and/or binding of the nativeFGF ligands, e.g., FGF19 and FGF21, to the KLB/FGFR1c complex. Incertain embodiments, an anti-KLB/anti-FGFR1 bispecific antibodydisclosed herein refers to an antibody that does not block the bindingand/or activity of native FGF ligands to an FGF receptor in the absenceof KLB. For example, and not by way of limitation, ananti-KLB/anti-FGFR1 bispecific antibody of the present disclosure doesnot block the interaction of native FGF ligands with the FGFR1/KLAcomplex or FGFR1 alone. In certain embodiments, an anti-KLB/anti-FGFR1bispecific antibody disclosed herein refers to an antibody that does notblock the binding and/or activity of native FGF ligands to KLB in theabsence of FGFR1. For example, and not by way of limitation, ananti-KLB/anti-FGFR1 bispecific antibody of the present disclosure doesnot block the interaction of native FGF ligands with the FGFR4/KLBcomplex, the FGFR2c/KLB complex and/or the FGFR3c/KLB complex.

In certain embodiments, anti-KLB antibodies, anti-FGFR1c and/oranti-KLB/anti-FGFR1, e.g., anti-KLB/anti-FGFR1c, bispecific antibodiesfor use in the disclosed methods can be labeled. Labels include, but arenot limited to, labels or moieties that are detected directly, such asfluorescent, chromophoric, electron-dense, chemiluminescent, andradioactive labels, as well as moieties, such as enzymes or ligands,that are detected indirectly, e.g., through an enzymatic reaction ormolecular interaction. Non-limiting examples of labels include theradioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I, fluorophores such as rareearth chelates or fluorescein and its derivatives, rhodamine and itsderivatives, dansyl, umbelliferone, luciferases, e.g., fireflyluciferase and bacterial luciferase (see U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP),alkaline phosphatase, 0-galactosidase, glucoamylase, lysozyme,saccharide oxidases, e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricaseand xanthine oxidase, coupled with an enzyme that employs hydrogenperoxide to oxidize a dye precursor such as HRP, lactoperoxidase, ormicroperoxidase, biotin/avidin, spin labels, bacteriophage labels,stable free radicals, and the like.

B. Therapeutic Methods

In certain embodiments, one or more antibodies of the presentlydisclosed subject matter can be used for treating a disease and/ordisorder in a subject. For example, but not by way of limitation, thedisease can be a metabolic disorder. Non-limiting examples of metabolicdisorders include polycystic ovary syndrome (PCOS), metabolic syndrome(MetS), obesity, non-alcoholic steatohepatitis (NASH), non-alcoholicfatty liver disease (NAFLD), hyperlipidemia, hypertension, type 2diabetes, non-type 2 diabetes, type 1 diabetes, latent autoimmunediabetes (LAD) and maturity onset diabetes of the young (MODY). Incertain embodiments, the metabolic disorder is type 2 diabetes. Incertain embodiments, the metabolic disorder is obesity.

In certain embodiments, one or more antibodies of the presentlydisclosed subject matter can be used to treat Bardet-Biedl syndrome,Prader-Willi syndrome, Alstrom syndrome, Cohen syndrome, Albright'shereditary osteodystrophy (pseudohypoparathyroidism), Carpentersyndrome, MOMO syndrome, Rubinstein-Taybi syndrome, fragile X syndromeand Börjeson-Forssman-Lehman syndrome. In certain embodiments, one ormore antibodies of the presently disclosed subject matter can be used totreat aging and related diseases such as Alzheimer's disease,Parkinson's disease and ALS.

In certain embodiments, one or more antibodies of the presentlydisclosed subject matter can be used to treat heart disease, stroke,heart attacks, hyperinsulinemia, high blood pressure, coronary-arterydisease, migraines or headaches directly related to obesity or cranialhypertension, congestive heart failure, neoplasia, dyslipidemia, anemia,gallbladder disease, osteoarthritis, degenerative arthritis,degenerative disc, degenerative joint disease, joint replacement,accelerated degenerative joint disease, asthma, repeated pneumonia,repeated pleurisy, repeated bronchitis, lung restriction,gastroesophageal reflex (gerd), excess facial and body hair (hirsutism),rashes, chronic skin infections, excess sweating, frequent yeastinfections, urinary stress incontinence, menstrual irregularity,hormonal abnormalities, polycystic ovaries, infertility, carcinoma(e.g., breast, colon and uterine cancer), sleep apnea, pseudotumorcerebri, depression, psychological/sexual dysfunction, socialdiscrimination and premature death.

In certain embodiments, the present disclosure provides an antibody foruse in a method of treatment. For example, and not by way of limitation,the present disclosure provides an antibody, e.g., ananti-KLB/anti-FGFR1 bispecific antibody, for use in a method of treatinga subject having a metabolic disorder, e.g., PCOS, MetS, obesity, NASH,NAFLD, hyperlipidemia, hypertension, type 2 diabetes, non-type 2diabetes, type 1 diabetes, LAD, MODY, and aging and related diseasessuch as Alzheimer's disease, Parkinson's disease and ALS, that includesadministering to the individual an effective amount of an antibody,disclosed herein. In certain embodiments, the present disclosureprovides an antibody, e.g., an anti-KLB/FGFR1 bispecific antibody, foruse in a method of treating a subject having a disease or disorderdescribed above, which includes administering to the individual aneffective amount of the antibody.

In certain embodiments, the method can further include administering tothe subject an effective amount of at least one additional therapeuticagent. Non-limiting examples of additional therapeutic agents, e.g., asecond therapeutic agent, are described below.

In certain embodiments, the present disclosure further provides a methodfor inducing weight loss comprising administering to an individual aneffective amount of one or more antibodies of the present disclosure,e.g., an anti-KLB/FGFR1 bispecific antibody.

An “individual,” “patient” or “subject,” as used interchangeably herein,refers to a mammal. Mammals include, but are not limited to,domesticated animals (e.g., cows, sheep, cats, dogs, and horses),primates (e.g., humans and non-human primates such as monkeys), rabbits,and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

The presently disclosed subject matter further provides an antibody,e.g., an anti-KLB/anti-FGFR1 bispecific antibody, for use in activatinga KLB/FGFR1c coreceptor complex, e.g., in a subject. For example, andnot by way of limitation, the anti-KLB/anti-FGFR1 bispecific antibodycan be an anti-KLB/anti-FGFR1c bispecific antibody. In certainembodiments, the present disclosure provides an antibody, e.g., ananti-KLB/anti-FGFR1 bispecific antibody, for use in a method ofactivating a KLB/FGFR1c coreceptor complex in a subject. In certainembodiments, the method includes administering to the subject aneffective of the antibody to activate a KLB/FGFR1c receptor complex.

Antibodies of the present disclosure can be used either alone or incombination with other agents in a therapy. For example, and not by wayof limitation, an antibody of the present disclosure can beco-administered with at least one additional therapeutic agent. Incertain embodiments, the second/additional therapeutic agent can includean anti-diabetic agent, an anti-obese agent or a medication formetabolic conditions such as, but not limited to, anti-hypertensivemedications and statins. Non-limiting examples of a second/additionaltherapeutic agent include metformin, pioglitazone, DPP4i, GLP1-analogs,sulfonylurea, insulin, Leptin-analogs and lorcaserin (e.g., BELVIQ®).

The present disclosure further provides for the use of an antibody,e.g., an anti-KLB/anti-FGFR1 bispecific antibody, in the manufacture orpreparation of a medicament. In certain embodiments, the medicament isfor treatment of a metabolic disorder, as disclosed above. In certainembodiments, the present disclosure provides the use of an antibody inthe manufacture of a medicament for treatment of obesity. In certainembodiments, the present disclosure provides the use of an antibody inthe manufacture of a medicament for treatment of type 2 diabetes. Incertain embodiments, the method further comprises administering to theindividual an effective amount of at least one additional therapeuticagent, e.g., as described herein. In certain embodiments, the medicamentis for activating a KLB/FGFR1c coreceptor complex. In certainembodiments, the medicament can be used in a method of activating aKLB/FGFR1c coreceptor complex in an individual comprising administeringto the individual an amount of the medicament effective to activate aKLB/FGFR1c receptor complex.

In certain embodiments, an antibody for use in the disclosed therapeuticmethods can be present in a pharmaceutical composition. In certainembodiments, the pharmaceutical composition can include apharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical composition can include one or more of the antibodies ofthe present disclosure.

Additionally or alternatively, the pharmaceutical composition caninclude a second therapeutic agent. When one or more of the disclosedantibodies are administered with another therapeutic agent, the one ormore antibodies and the other therapeutic agent can be administered ineither order or simultaneously. Such combination therapies noted aboveencompass combined administration (where two or more therapeutic agentsare included in the same or separate formulations), and separateadministration, in which case, administration of the antibody of thepresent disclosure can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent or agents. In oneembodiment, administration of an antibody of the present disclosure andadministration of an additional therapeutic agent occur within about onemonth, or within about one, two or three weeks, or within about one,two, three, four, five, or six days, of each other.

An antibody of the present disclosure (and any additional therapeuticagent) can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.,by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Antibodies of the present disclosure would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the present disclosure (when used alone or in combinationwith one or more other additional therapeutic agents) will depend on thetype of disease to be treated, the type of antibody, the severity andcourse of the disease, whether the antibody is administered forpreventive or therapeutic purposes, previous therapy, the patient'sclinical history and response to the antibody, and the discretion of theattending physician. In certain embodiments, an antibody of the presentdisclosure can be administered on an as needed basis. In certainembodiments, the antibody can be administered to the patient one time orover a series of treatments. For example, but not by way of limitation,the antibody and/or pharmaceutical formulation contains an antibody, asdisclosed herein, can be administered to a subject twice every day, onceevery day, once every two days, once every three days, once every fourdays, once every five days, once every six days, once a week, once everytwo weeks, once every three weeks, once every month, once every twomonths, once every three months, once every six months or once everyyear.

In certain embodiments, depending on the type and severity of thedisease, about 1 μg/kg to 15 mg/kg (e.g., 0.1 mg/kg-Omg/kg) of antibodycan be an initial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, or bycontinuous infusion. One typical daily dosage might range from about 1μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Incertain embodiments, the daily dosage can be greater than about 100mg/kg. In certain embodiments, dosage can be adjusted to achieve aplasma antibody concentration of 1-1000 μg/ml and in some methods 25-300μg/ml.

For repeated administrations over several days or longer, depending onthe condition, the treatment could generally be sustained until adesired suppression of disease symptoms occurs. One exemplary dosage ofthe antibody would be in the range from about 0.05 mg/kg to about 10mg/kg. In certain embodiments, one or more doses of about 0.5 mg/kg, 2.0mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can beadministered to the patient. Alternatively, antibody can be administeredas a sustained release formulation, in which case less frequentadministration is required. Dosage and frequency can vary based on thehalf-life of the antibody in the patient. In certain embodiments, suchdoses may be administered intermittently, e.g., every week or everythree weeks (e.g., such that the patient receives from about two toabout twenty, or, e.g., about six doses of the antibody). An initialhigher loading dose, followed by one or more lower doses may beadministered.

In certain embodiments, the method can further include monitoring thesubject and determining the effectiveness of the treatment. For example,the progress of this therapy can be easily monitored by conventionaltechniques and assays.

IV. Pharmaceutical Formulations

The presently disclosed subject matter further provides pharmaceuticalformulations containing one or more antibodies, as described herein,with a pharmaceutically acceptable carrier. In certain embodiments, thepharmaceutical compositions can include a combination of multiple (e.g.,two or more) antibodies and/or antigen-binding portions thereof of thepresently disclosed subject matter. In certain embodiments, apharmaceutical composition of the present disclosure can include one ormore anti-KLB/anti-FGFR1 bispecific antibodies.

In certain embodiments, the disclosed pharmaceutical formulations can beprepared by combining an antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Forexample, but not by way of limitation, lyophilized antibody formulationsare described in U.S. Pat. No. 6,267,958. In certain embodiments,aqueous antibody formulations can include those described in U.S. Pat.No. 6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

In certain embodiments, the antibody can be of a purity greater thanabout 80%, greater than about 90%, greater than about 91%, greater thanabout 92%, greater than about 93%, greater than about 94%, greater thanabout 95%, greater than about 96%, greater than about 97%, greater thanabout 98%, greater than about 99%, greater than about 99.1%, greaterthan about 99.2%, greater than about 99.3%, greater than about 99.4%,greater than about 99.5%, greater than about 99.6%, greater than about99.7%, greater than about 99.8% or greater than about 99.9%.

Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

The carrier can be suitable for intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion). Depending on the route of administration, theactive compound, i.e., an anti-KLB/anti-FGFR1 bispecific antibody, canbe coated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

Pharmaceutical compositions of the present disclosure also can beadministered in combination therapy, i.e., combined with other agents.In certain embodiments, pharmaceutical compositions disclosed herein canalso contain more than one active ingredients as necessary for theparticular indication being treated, for example, those withcomplementary activities that do not adversely affect each other. Incertain embodiments, the pharmaceutical formulation can include a secondactive ingredient for treating the same disease treated by the firsttherapeutic. Such active ingredients are suitably present in combinationin amounts that are effective for the purpose intended. For example, andnot by way of limitation, the formulation of the present disclosure canalso contain more than one active ingredients as necessary for theparticular indication being treated, preferably those with complementaryactivities that do not adversely affect each other. For example, it maybe desirable to further provide a second therapeutic useful fortreatment of the same disease. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

A composition of the present disclosure can be administered by a varietyof methods known in the art. The route and/or mode of administrationvary depending upon the desired results. The active compounds can beprepared with carriers that protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are described by e.g., Sustained and ControlledRelease Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc.,New York, 1978. In certain embodiments, the pharmaceutical compositionsare manufactured under Good Manufacturing Practice (GMP) conditions ofthe U.S. Food and Drug Administration.

Sustained-release preparations containing a disclosed antibody can alsobe prepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theantibody, which matrices are in the form of shaped articles, e.g. films,or microcapsules. In certain embodiments, active ingredients can beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

To administer an antibody of the present disclosure by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present disclosure is contemplated. Supplementary active compoundscan also be incorporated into the compositions.

Therapeutic compositions typically must be sterile, substantiallyisotonic, and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion,liposome, or other ordered structure suitable to high drugconcentration. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and liquid polyethylene glycol, and the like), andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. In many cases, it is preferable to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be brought about by including in the composition anagent that delays absorption, for example, monostearate salts andgelatin.

Sterile injectable solutions can be prepared by incorporating one ormore disclosed antibodies in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by sterilization microfiltration, e.g., by filtrationthrough sterile filtration membranes. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Therapeutic compositions can also be administered with medical devicesknown in the art. For example, a therapeutic composition of the presentdisclosure can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in, e.g., U.S. Pat. Nos.5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or4,596,556. Examples of implants and modules useful in the presentdisclosure include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known.

For the therapeutic compositions, formulations of the present disclosureinclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations can conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofantibody, which can be combined with a carrier material to produce asingle dosage form, vary depending upon the subject being treated, andthe particular mode of administration. The amount of the antibody whichcan be combined with a carrier material to produce a single dosage formgenerally be that amount of the composition which produces a therapeuticeffect. Generally, out of one hundred percent, this amount range fromabout 0.01 percent to about ninety-nine percent of active ingredient,from about 0.1 percent to about 70 percent, or from about 1 percent toabout 30 percent.

Dosage forms for the topical or transdermal administration ofcompositions of the present disclosure include powders, sprays,ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”mean modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.

These pharmaceutical compositions can also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form can be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In certain embodiments, when the antibodies of the present disclosureare administered as pharmaceuticals, to humans and animals, they can begiven alone or as a pharmaceutical composition containing, for example,from about 0.01% to about 99.5% (or about 0.1 to about 90%) of anantibody, described herein, in combination with a pharmaceuticallyacceptable carrier.

V. Articles of Manufacture

The presently disclosed subject matter further relates articles ofmanufacture containing materials useful for the treatment, preventionand/or diagnosis of the disorders described above.

In certain embodiments, the article of manufacture includes a containerand a label or package insert on or associated with the container.Non-limiting examples of suitable containers include bottles, vials,syringes, IV solution bags, etc. The containers can be formed from avariety of materials such as glass or plastic. The container can hold acomposition which is by itself or combined with another compositioneffective for treating, preventing and/or diagnosing the condition andmay have a sterile access port (for example, the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle).

In certain embodiments, at least one active agent in the composition isan antibody of the presently disclosed subject matter. The label orpackage insert can indicate that the composition is used for treatingthe condition of choice.

In certain embodiments, the article of manufacture can comprise (a) afirst container with a composition contained therein, wherein thecomposition comprises an antibody of the present disclosure; and (b) asecond container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. In certain embodiments, the article of manufacture can furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition.

Alternatively, or additionally, the article of manufacture can furtheran additional container, e.g., a second or third container, including apharmaceutically-acceptable buffer, such as, but not limited to,bacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. The article of manufacture caninclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, and syringes.

The following examples are merely illustrative of the presentlydisclosed subject matter and should not be considered as limitations inany way.

EXAMPLES Example 1: Characterization of Anti-FGFR1 Agonist Antibodies

Three phage-derived anti-FGFR1 antibodies, YW182.2 (also referred toherein as “R1MAb1”), YW182.3 (also referred to herein as “R1MAb2”), andYW182.5 (also referred to herein as “R1MAb3”) were previously described(WO 2012/158704, incorporated by reference herein)). Each of the threeantibodies acts as a potent FGFR1-selective agonist and exhibitedinsulin-sensitizing properties in mice.

To further understand this agonistic activity, the ability of Fabfragments of these antibodies to agonize FGFR1c was tested. HEK293 cellswere cultured in Dulbecco's Modified Eagle Medium (DMEM)+10% fetalbovine serum (FBS), and transiently-transfected with expression vectorsencoding Renilla luciferase (pRL-SV40, Promega), FGFR1c, atranscriptional activator (pFA2-Elkl or pFA2-CREB, Stratagene), and afirefly luciferase reporter driven by GAL4 binding sites (pFR-luc,Stratagene), using FUGENE® HD Transfection Reagent (Roche). On the nextday, the transfected cells were cultured for an additional 6-8 h inserum free media and YW182.5 IgG and each of YW182.2, YW182.3 andYW182.5 were tested at increasing concentrations. The cellularluciferase activity was determined using DUAL-GLO® Luciferase AssaySystem (Promega) and ENVISION® Multilabel Reader (PerkinElmer). Fireflyluciferase activity was normalized to the co-expressed Renillaluciferase activity. Surprisingly, YW182.2 Fab, but not YW182.3 Fab orYW182.5 Fab, exhibited agonistic activity (FIG. 1A).

FIG. 1B depicts the binding competition experiments that were performedto explore the basis for the difference in FGFR1 activation by anYW182.2 Fab and an YW182.3 Fab. YW182.2 was further characterized incomparison to YW182.3, which has high affinity, and in comparison to thelower affinity anti-FGFR1 antibody, YW182.5. Both YW182.2 and YW182.3competed with YW182.5 for the binding to the FGFR1 extracellular domain(ECD), indicating that all 3 antibodies recognize an overlapping regionof FGFR1. However, as shown in FIG. 1B, the relative affinity of YW182.5was significantly weaker (IC₅₀>30 fold) than that of YW182.2 andYW182.3.

FIG. 2A depicts the binding affinities of the anti-FGFR1 antibodies,YW182.2 and YW182.3, for FGFR1b and FGFR1c. The affinity of theanti-FGFR1 antibodies was determined to assess whether differences inaffinity of the anti-FGFR1 antibodies explain the differences observedin agonistic activity. The binding affinities of the Fabs to FGFR1b orFGFR1c using a BIACORE® T100 instrument was performed as described inLiang et al. J. Mol. Biol. 366(3): 815-29 (2007), with the followingmodifications. Mouse anti-human Fe antibody was first coated on aBIAcore carboxymethylated dextran CM5 chip using direct coupling to freeamino groups following a procedure described by the manufacturer.YW182.2 or YW182.3 was then captured on CM5 biosensor chips to achieveapproximately 200 response units (RU). Binding measurements wereperformed using a running buffer composed of 10 mM HEPES pH 7.4, 150 mMNaCl, 0.005% surfactant P20 (HBS-P buffer). A 2 fold dilution series ofFGFR1c ECD-His protein was injected in a range of 1.5-50 nM in HBS Pbuffer at a flow rate of 30 μL/minute at 25° C. Association rates(K_(on), per mol/s) and dissociation rates (K_(off), per s) werecalculated using a simple one-one Langmuir binding model (BiacoreEvaluation Software version 3.2). The equilibrium dissociation constant(K_(d), per mol) was calculated as the ratio of K_(off)/K_(on). As shownin FIG. 2A, the affinities of YW182.2 and YW182.3 were observed to bevery similar, indicating that the affinity could not explain thedifference between the agonistic activities of the two antibodies.

FIG. 2B shows the ability of YW182.5 (R1MAb3) to specifically interactwith FGFR1. Like YW182.2 and YW182.3, YW182.5 showed specific binding toFGFR1 by ELISA (FIG. 2B).

FIG. 2C depicts the agonistic activity of YW182.5 for various FGFRs inL6 cells using a GAL-ELK1 (ETS-like transcription factor 1) basedluciferase assay. For the luciferase assay, HEK293T or rat L6 cells weretransiently transfected with expression vectors encoding appropriatereceptors under the CMV-promoter, Renilla luciferase (pRL-SV40,Promega), GAL-ELK1 transcriptional activator fusion (pFA2-ELK1,Agilent), and a firefly luciferase reporter driven by GAL4 binding sites(pFR-luc, Agilent), using FuGENE HD Transfection Reagent (Promega). Onthe next day, the transfected cells were cultured for an additional 6-8hours in serum free DMEM-based media containing appropriate proteinligands at various concentrations. The cellular luciferase activity wasdetermined using Dual-Glo Luciferase Assay System (Promega) and EnVisionMultilabel Reader (PerkinElmer). Firefly luciferase activity wasnormalized to the co-expressed Renilla luciferase activity, and shown asmeans±SEM. Similar to YW182.2 and YW182.3, YW182.5 acted as a specificagonist for FGFR1 in L6 cells (FIG. 2C).

The agonistic activity of YW182.5 was further tested in HEK293 cellsusing the GAL-ELK1-based luciferase assay described above. As shown inFIG. 2D, YW182.5 also acted as a specific agonist for FGFR1c in theGAL-ELK1 based luciferase assay in HEK293 cells.

FIG. 2E shows the effect of YW182.5 on blood glucose levels in adiabetic mouse model. To determine blood glucose levels, mice werepurchased from Jackson Laboratory and maintained in a pathogen-freeanimal facility at 21° C. under standard 12 h light/12 h dark cycle withaccess to chow (LABDIET® 5010) and water ad libitum. db/db mice inC57BLKS/J background were females and other mice were all males. Forhigh-fat diet feeding, a high fat, high carbohydrate diet (Harlan TekladTD.03584, 58.4% calories from fat) was used. Serum inorganic phosphateand calcium levels were determined by COBAS INTEGRA® 400 ChemistryAnalyzer (Roche). Serum FGF23 levels were determined by ELISA(Immutopics). Blood glucose levels were determined by CONTOUR® glucosemeter (Bayer). For hepatic lipid analysis, triglyceride quantificationkit (MBL International) was used. Serum total cholesterol,triglycerides, p-hydroxybutyrate (Thermo DMA) and free fatty acid(Roche) were determined by colorimetric assays. ELISA was used todetermine serum insulin levels (Crystal Chem), serum FGF23 (Immutopics),serum mouse HMW adiponectin (Alpco) and serum monkey HMW adiponectin(R&D systems). Corticosterone was measured by radioimmunoassay(Vanderbilt Hormone Assay & Analytical Services Core). All the mice usedfor injection were around 2-4 months old, except klb-deficient mice,which were used in certain experiments at 7-8 months old. In a similarmanner to YW182.2 and YW182.3, YW182.5 normalized blood glucose levelswhen injected into diabetic ob/ob mice (FIG. 2E).

Example 2: Epitope Mapping of Anti-FGFR1 Antibodies

The FGFR1 ECD consists of three Ig-like domains called D1 to D3. Asshown in FIG. 1C, two non-overlapping peptides (P26: KLHAVPAAKTVKFKCP(SEQ ID NO: 143) and P28: FKPDHRIGGYKVRY (SEQ ID NO: 144) are presentwithin the D2 domain of FGFR1 and were previously identified to bind toboth YW182.2 and YW182.3 (WO 2012/158704, incorporated by referenceherein).

To identify which residues in these peptides are most responsible forantibody binding, full-length FGFR1 proteins with various alaninesubstitutions within the identified epitope regions were expressed inHEK293 cells and tested for antibody binding by western blot. As shownin FIG. 1D, alanine substitution in K175, K177, Y205, R208, eliminatedbinding of YW182.2 and YW182.5, without affecting expression as probedby anti-FGFR1 against D1 domain (anti-D1). Binding of YW182.3 wasabolished by R208A, but not by the K175, K177, or Y205 substitutions.

The ability of the antibodies to activate the alanine substitutionmutants of FGFR1 in vivo was tested using the GAL-ELK1 assay describedabove. It was found that activation correlated well with the bindingproperties of these mutants to each anti-FGFR1 antibody (FIG. 1E). Theseresults suggest that a similar set of amino acids within the D2 domainare required for YW182.2 and YW182.5 binding with albeit differentaffinity, whereas a distinct set of amino acids in the same region isimportant for YW182.3 binding.

Crystal structures of 2:2 FGFR ECD/FGF complex have previously beendescribed (Plotnikov et al. Cell 98(5): 641-50 (1999)). In the 2:2homodimeric FGFR1c ECD/FGF2 structures, one D2 domain interacts withanother D2 domain, with each FGF2 binds to both D2 domains from twosides to stabilize the D2 dimer (FIG. 1F). In these structures, thealanine substitutions important for YW182.2 and YW182.5 binding (K175,K177, Y205, and R208) are situated inside of the D2 dimer. Since YW182.2Fab acts as an agonist, this suggested that YW182.2 Fab may bind to twoD2 domains simultaneously from the side to stabilize the D2 dimer,essentially acting as a molecular mimetic of FGF ligands. Based on thealanine substitution analysis, YW182.5 Fab might bind similarly exceptthat the affinity is much lower than YW182.2 Fab.

Example 3: Isolation and Characterization of Anti-KLB Antibodies

Balb/c mice were immunized with HEK293 cells stably expressing hFGFR1cand hKLB protein. Spleens were harvested after 12 weeks and hybridomaswere generated. Anti-hKLB antibody producing hybridomas were identifiedby FACS analysis using the HEK293 cells used for immunization. Briefly,293 cells expressing hKLB alone, hFGFR1 alone, or both, were stainedwith diluted hybridoma supernatant and PE-conjugated goat anti-mouse IgGantibody (Jackson Labs) is FACS buffer (0.5% BSA in PBS). The same FACSbuffer was used to wash the stained cells. Stained cells were analyzedby FACScan (Becton Dickinson) and FlowJo FACS analysis software (TreeStar). cDNA encoding the IgG heavy chain and light chain were clonedinto expression vectors. All the recombinant monoclonal antibodymolecules were produced in transiently transfected Chinese hamster ovary(CHO) cells and purified using conventional column chromatography.

Approximately 20 different hybridomas producing anti-KLB antibodies wereidentified. The CDR light chain and heavy chain sequences for 16 ofthese anti-KLB antibodies are shown in Tables 2 and 3. The light chainsequences of 16 of these anti-KLB antibodies along with 8C5 are shown inFIG. 3A (11F1, 6D12, 11D4, 8E1, 46C3, 8H7, 21H3, 25F7, 14E6, 14C6, 24A1,5F8, 6C1, 12A1, 12B8, 14C10 and 8C5; SEQ ID NOs: 111-127, respectively).

The heavy chain sequences of 16 of these anti-KLB antibodies along with8C5 are shown in FIG. 3B (11F1, 6D12, 11D4, 8E1, 46C3, 8H7, 21H3, 25F7,14E6, 14C6, 24A1, 5F8, 6C1, 12A1, 12B8, 14C10 and 8C5; SEQ ID NOs:94-110, respectively).

TABLE 2 CDR H sequences for murine anti-KLB monoclonal antibodies.Antibody CDR H1 CDR H2 CDR H3 11F1 SYGIS TVSSGGRYTYYPDSVKG GGDGYALDY(SEQ ID NO: 1) (SEQ ID NO: 16) (SEQ ID NO: 32) 6D12 DYYMNWIDPENDDTIYDPKFQG FTTVFAY (SEQ ID NO: 2) (SEQ ID NO: 17) (SEQ ID NO: 33)11D4 NYGVS VIWGDGSINYHSALIS THDWFDY (SEQ ID NO: 3) (SEQ ID NO: 18) (SEQID NO: 34) 8E1 DTYMN RIDPSNGNAKYDPKFQG RALGNGYALGY (SEQ ID NO: 4) (SEQID NO: 19) (SEQ ID NO: 35) 46C3 DTYIH RIDPANGNTKYDPKFQD GTSYSWFAY (SEQID NO: 5) (SEQ ID NO: 20) (SEQ ID NO: 36) 8H7 SYWIH EIDPSVSNSNYNQKFKGLGVMVYGSSPFWFAY (SEQ ID NO: 6) (SEQ ID NO: 21) (SEQ ID NO: 37) 21H3SYWIH EIDPSVSNSNYNQKFKG LGVMVYGSSPFWFAY (SEQ ID NO: 6) (SEQ ID NO: 21)(SEQ ID NO: 37) 25F7 DTFTH RIDPSNGNTKYDPKFQG RALGNGYAMDY (SEQ ID NO: 7)(SEQ ID NO: 22) (SEQ ID NO: 38) 14E6 EYTMN (SEQ ID GINPNNGETSYNQKFKGKTTNY NO: 8) (SEQ ID NO: 23) (SEQ ID NO: 39) 14C6 SYWIEEIFPGGGSTIYNENFRD RGYYDAAWFDY (SEQ ID NO: 9) (SEQ ID NO: 24) (SEQ ID NO:40) 24A1 DYEMH AIWPENADSVYNQKFKG EGGNY (SEQ ID NO: 10) (SEQ ID NO: 25)(SEQ ID NO: 41) 5F8 DTYIH RIDPANGNTKYDPKFQG SGNYGAMDY (SEQ ID NO: 11)(SEQ ID NO: 26) (SEQ ID NO: 42) 6C1 SYWIE EILPGSDSTKYVEKFKV GGYHYPGWLVY(SEQ ID NO: 9) (SEQ ID NO: 27) (SEQ ID NO: 43) 12A11 RYWMSEISPDSSTINYTPSLKD PSPALDY (SEQ ID NO: 12) (SEQ ID NO: 28) (SEQ ID NO:44) 12B8 NYGMN WIDTDTGEATYTDDFKG EEYGLFGFPY (SEQ ID NO: 13) (SEQ ID NO:29) (SEQ ID NO: 45) 14C10 TSAMGIG HIWWDDDKRYNPALKS IDGIYDGSFYAMDY (SEQID NO: 14) (SEQ ID NO: 30) (SEQ ID NO: 46) 8C5 TYGVH VIWSGGSTDYNAAFISDYGSTYVDAIDY (SEQ ID NO: 15) (SEQ ID NO: 31) (SEQ ID NO: 47)

TABLE 3 CDR L sequences for murine anti-KLB monoclonal antibodies.Antibody CDR L1 CDR L2 CDR L3 11F1 SASQVISNYLN FTSSLRS QQYSKLPWT (SEQ IDNO: 48) (SEQ ID NO: 63) (SEQ ID NO: 79) 6D12 SASSSGRYTF DTSKLASFQGTGYPLT (SEQ ID NO: 49) (SEQ ID NO: 64) (SEQ ID NO: 80) 11D4RASQDISNYFN YTSRLQS HQVRTLPWT (SEQ ID NO: 50) (SEQ ID NO: 65) (SEQ IDNO: 81) 8E1 KASDHINNWLA GTTNLET QQYWNTPFT (SEQ ID NO: 51) (SEQ ID NO:66) (SEQ ID NO: 82) 46C3 RSSQNIVHSDGNTYLE KVSNRFS FQGSHVLT (SEQ ID NO:52) (SEQ ID NO: 67) (SEQ ID NO: 83) 8H7 KASQFVSDAVA SASYRYT QQHYIVPYT(SEQ ID NO: 53) (SEQ ID NO: 68) (SEQ ID NO: 84) 21H3 KASQFVSDAVA SASYRYTQQHYIVPYT (SEQ ID NO: 53) (SEQ ID NO: 68) (SEQ ID NO: 84) 25F7KASDHINNWLA GASNLET QQYWNTPFT (SEQ ID NO: 51) (SEQ ID NO: 69) (SEQ IDNO: 82) 14E6 RASQEISGYLS AASTLDS LQYGSYPWT (SEQ ID NO: 54) (SEQ ID NO:70) (SEQ ID NO: 85) 14C6 SASSSLSSSYLY GASNLAS HQWSSYPLT (SEQ ID NO: 55)(SEQ ID NO: 71) (SEQ ID NO: 86) 24A1 KSSQSLLNSGNQKNSLA LASTRES QQHHSTPYT(SEQ ID NO: 56) (SEQ ID NO: 72) (SEQ ID NO: 87) 5F8 RASSSVNHMY YTSTLAPQQFTISPSMYT (SEQ ID NO: 57) (SEQ ID NO: 73) (SEQ ID NO: 88) 6C1KASQNVDSYVA SASYRFS QQYNISPYT (SEQ ID NO: 58) (SEQ ID NO: 74) (SEQ IDNO: 89) 12A11 RASQSISDYVY YASQSIS QNGHNFPYT (SEQ ID NO: 59) (SEQ ID NO:75) (SEQ ID NO: 90) 12B8 KASEDIYNRLA AATSLET QQYWSNPLT (SEQ ID NO: 60)(SEQ ID NO: 76) (SEQ ID NO: 91) 14C10 RASESVDSYGNSFMH RASNLES QQSNEDYT(SEQ ID NO: 61) (SEQ ID NO: 77) (SEQ ID NO: 92) 8C5 RASESVESYGNRYMTRAANLQS QQSNEDPWT (SEQ ID NO: 62) (SEQ ID NO: 78) (SEQ ID NO: 93)

Most of the hybridoma-derived anti-KLB antibodies along with onephage-derived antibody (designate Ph #5, which was obtained by phagepanning using recombinant hKLB-ECD-HIS protein (R&D Systems)) wereranked based on the median shift observed in the FACS plot at 0.8 μg/mlmeasuring binding of the antibodies to 293 cells expressing hKLB (FIG. 4).

In addition, some of the antibodies were ranked by ELISA. For theseexperiments, anti-KLB antibodies that were chimeric recombinant IgG withmurine variable regions and hIgG1 constant regions were used to measurebinding to hKLB-ECD-HIS protein. The relative binding of the antibodiestested were similar except for 14E6, which appeared to bind better underthe ELISA conditions than in the FACS analysis (FIG. 5 ).

Example 4: KLB Binding of Anti-KLB Antibodies

To test competition between various anti-KLB antibodies, ELISA was used.In some experiments, IgG antibodies purified from hybridoma supernatantscorresponding to 6D12, 8C5, and 11F1 were biotinylated using EZ-linkNHS-PEO Solid Phase Biotinylation Kit (Pierce). Binding to KLB-ECD-HISprotein was tested using HRP-conjugated streptavidin in the presence ofvarious concentrations of hybridoma-derived anti-KLB. In someexperiments, binding of recombinant human IgG to KLB-ECD-HIS protein wastested using HRP-conjugated anti-human IgG (Jackson ImmunoResearch Inc.)in the presence of various concentrations of hybridoma-derived anti-KLB.It was observed that none of 11F1, 11D4, 8E1 and 46C3 compete with 6D12for binding (others were not tested against 6D12). Anti-KLB antibodies14E6 and 12A11 compete for binding with 8C5, but 11D4 and 14C10 do not(others were not tested against 8C5), and 11D4 competes for binding with11F1, but 6D12, 8E1, and 46C3 (others were not tested against 11F1).

Example 5: Cross-Species Crossreactivity of Anti-KLB Antibodies

Species cross reactivity of the disclosed anti-KLB antibodies wereanalyzed by FACS analysis using KLB cDNA from mouse, rat, rabbit,cynomolgus monkey and rhesus monkey cloned into pRK mammalian expressionvectors transiently transfected into HEK293T cells. The KLBextracellular domain polypeptide sequences that were expressed are asfollows:

Mouse: (SEQ ID NO: 158)FSGDGKAIWDKKQYVSPVNPSQLFLYDTFPKNFSWGVGTGAFQVEGSWKTDGRGPSIWDRYVYSHLRGVNGTDRSTDSYIFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAQGLRYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLENDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTDNMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTGAMIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDDPQILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIRVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLKESTPDMKGRFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKVNRQVLRYYRCVVSEGLKLGVFPMVTLYHPTHSHLGLPLPLLSSGGWLNMNTAKAFQDYAELCFRELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHCDWAEPANPFVDSHWKAAERFLQFEIAWFADPLFKTGDYPSVMKEYIASKNQRGLSSSVLPRFTAKESRLVKGTVDFYALNHFTTRFVIHKQLNTNRSVADRDVQFLQDITRLSSPSRLAVTPWGVRKLLAWIRRNYRDRDIYITANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFRAKSSVQFYSKLISSSGLPAENRSPACGQPAEDTDC TICSFLV.Rat (+N-ter FLAG): (SEQ ID NO: 147)DYKDDDDKLEFSGDGKAIWDKKQYVSPVNPGQLFLYDTFPKNFSWGVGTGAFQVEGSWKADGRGPSIWDRYVDSHLRGVNSTDRSTDSYVFLEKDLLALDFLGVSFYQFSISWPRLFPNGTVAAVNAKGLQYYRALLDSLVLRNIEPIVTLYHWDLPLTLQEEYGGWKNATMIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGFGTGMHAPGEKGNLTAVYTVGHNLIKAHSKVWHNYDKNFRPHQKGWLSITLGSHWIEPNRTENMEDVINCQHSMSSVLGWFANPIHGDGDYPEFMKTSSVIPEFSEAEKEEVRGTADFFAFSFGPNNFRPSNTVVKMGQNVSLNLRQVLNWIKLEYDNPRILISENGWFTDSYIKTEDTTAIYMMKNFLNQVLQAIKFDEIQVFGYTAWTLLDGFEWQDAYTTRRGLFYVDFNSEQKERKPKSSAHYYKQIIQDNGFPLQESTPDMKGQFPCDFSWGVTESVLKPEFTVSSPQFTDPHLYVWNVTGNRLLYRVEGVRLKTRPSQCTDYVSIKKRVEMLAKMKVTHYQFALDWTSILPTGNLSKINRQVLRYYRCVVSEGLKLGISPMVTLYHPTHSHLGLPMPLLSSGGWLNTNTAKAFQDYAGLCFKELGDLVKLWITINEPNRLSDMYNRTSNDTYRAAHNLMIAHAQVWHLYDRQYRPVQHGAVSLSLHSDWAEPANPYVESHWKAAERFLQFEIAWFADPLFKTGDYPLAMKEYIASKKQRGLSSSVLPRFTLKESRLVKGTIDFYALNHFTTRFVIHKQLNTNCSVADRDVQFLQDITRLSSPSRLAVTPWGMRKLLGWIRRNYRDMDIYVTANGIDDLALEDDQIRKYYLEKYVQEALKAYLIDKVKIKGYYAFKLTEEKSKPRFGFFTSDFKAKSSVQFYSKLISSSGFSSENRSPA CGQPPEDTECAICSFLT.Rabbit (+N-ter FLAG): (SEQ ID NO: 148)DYKDDDDKLDFPGDGRAVWSQNPNLSPVNESQLFLYDTFPKNFFWGVGTGAFQVEGSWKKDGKGLSVWDHFIATHLNVSSRDGSSDSYIFLEKDLSALDFLGVSFYQFSISWPRLFPDGTVAVANAKGLQYYNRLLDSLLLRNIEPVVTLYHWDLPWALQEKYGGWKNETLIDLFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGYGTGLHAPGEKGNVAAVYTVGHNLLKAHSKVWHNYNRNFRPHQKGWLSITLGSHWIEPNRAESIVDILKCQQSMVSVLGWFANPIHGDGDYPEVMTKKLLSVLPAFSEAEKNEVRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLRQVLNWIKLEYGNPRILIAENGWFTDSYVQTEDTTAIYMMKNFLNQVLQAIRLDGVRVFGYTAWSLLDGFEWQDAYNTRRGLFYVDFNSEQRERRPKSSAHYYKQVIGENGFTLREATPDLQGQFPCDFSWGVTESVLKPESVASSPQFSDPHLYVWNATGNRMLHRVEGVRLKTRPAQCTDFITIKKQLEMLARMKVTHFRFALDWASVLPTGNLSEVNRQALRYYRCVVTEGLKLNISPMVTLYYPTHAHLGLPAPLLHSGGWLDPSTAKAFRDYAGLCFRELGDLVKLWITINEPNRLSDVYNRTSNDTYQAAHNLLIAHAIVWHLYDRQYRPSQRGALSLSLHSDWAEPANPYVASHWQAAERFLQFEIAWFAEPLFKTGDYPVAMREYIASKTRRGLSSSVLPRFSDAERRLVKGAADFYALNHFTTRFVMHEQQNGSRYDSDRDVQFLQDITRLASPSRLAVMPWGEGKLLRWMRNNYGDLDVYITANGIDDQALQNDQLRQYYLEKYVQEALKAYLIDKIKIKGYYAFKLTEEKSKPRFGFFTSDFKAKSSIQFYNKLITSNGFPSENGGP RCNQTQGNPECTVCLLLL.Cynomolgus monkey (+N-ter FLAG): (SEQ ID NO: 149)DYKDDDDKLEFSGDGRAVWSKNPNFTPVNESQLFLYDTFPKNFFWGVGTGALQVEGSWKKDGKGPSIWDHFVHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYNTLLDSLVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDILKCQQSMVSVLGWFASPIHGDGDYPEGMKKKLLSILPLFSEAEKNEVRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPRILIAENGWFTDSHVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKEATPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPYLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHAGGWLNPSTVEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLEKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKMISSSGFPSENSS SRCSQTQKNTECTVCLFLA.Rhesus monkey (+N-ter FLAG): (SEQ ID NO: 150)DYKDDDDKLEFSGDGRAVWSKNPNFTPVNESQLFLYDTFPKNFFWGVGTGALQVEGSWKKDGKGPSIWDHFVHTHLKNVSSTNGSSDSYIFLEKDLSALDFIGVSFYQFSISWPRLFPDGIVTVANAKGLQYYNALLDSLVLRNIEPIVTLYHWDLPLALQEKYGGWKNDTIIDIFNDYATYCFQTFGDRVKYWITIHNPYLVAWHGYGTGMHAPGEKGNLAAVYTVGHNLIKAHSKVWHNYNTHFRPHQKGWLSITLGSHWIEPNRSENTMDILKCQQSMVSVLGWFANPIHGDGDYPEGMKKKLLSILPLFSEAEKNEVRGTADFFAFSFGPNNFKPLNTMAKMGQNVSLNLREALNWIKLEYNNPQILIAENGWFTDSHVKTEDTTAIYMMKNFLSQVLQAIRLDEIRVFGYTAWSLLDGFEWQDAYTIRRGLFYVDFNSKQKERKPKSSAHYYKQIIRENGFSLKEATPDVQGQFPCDFSWGVTESVLKPESVASSPQFSDPYLYVWNATGNRLLHRVEGVRLKTRPAQCTDFVNIKKQLEMLARMKVTHYRFALDWASVLPTGNLSAVNRQALRYYRCVVSEGLKLGISAMVTLYYPTHAHLGLPEPLLHAGGWLNPSTVEAFQAYAGLCFQELGDLVKLWITINEPNRLSDIYNRSGNDTYGAAHNLLVAHALAWRLYDRQFRPSQRGAVSLSLHADWAEPANPYADSHWRAAERFLQFEIAWFAEPLFKTGDYPAAMREYIASKHRRGLSSSALPRLTEAERRLLKGTVDFCALNHFTTRFVMHEQLAGSRYDSDRDIQFLQDITRLSSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITASGIDDQALEDDRLRKYYLEKYLQEVLKAYLIDKVRIKGYYAFKLAEEKSKPRFGFFTSDFKAKSSIQFYNKMISSSGFPSENSS SRCSQTQKNTECTVCLFLV.

As shown in Table 4, most antibodies, e.g., 6D12, 11D4 and 8E1, werefound to bind to KLB from rabbit, cynomolgus monkey and rhesus monkeyand about half of the anti-KLB antibodies, e.g., 8C5, 14E6 and 14C6,were found to bind to mouse and rat KLB.

TABLE 4 Binding of murine anti-KLB antibodies to KLB from differentspecies. Anti-KLB Cynomolgus Rhesus Antibody Mouse Rat Rabbit MonkeyMonkey 11F1 no no no YES YES 6D12 no no YES YES YES 11D4 no no YES YESYES 8E1 no no YES YES YES 46C3 no no YES no no 8H7 Weak no YES YES YES21H3 Weak no YES YES YES 25F7 no no Weak YES YES 8C5 YES YES no YES YES14E6 YES YES YES YES YES 14C6 YES YES YES YES YES 24A1 YES YES YES YESYES 5F8 no no YES YES YES 6C1 no no YES YES YES 12A11 Weak no YES YESYES 12B8 no no YES YES YES 14C10 no no YES YES YES

Example 6: Epitope Mapping of Anti-KLB Antibodies

To determine whether the anti-KLB antibodies do not bind theextracellular domain (ECD) of human alpha-Klotho (hKLA), a constructhaving the following sequence was used:

Predicted polypeptide sequence expressed (withC-terminal (intracellular) FLAG): (SEQ ID NO: 151)EPGDGAQTWARFSRPPAPEAAGLFQGTFPDGFLWAVGSAAYQTEGGWQQHGKGASIWDTFTHHPLAPPGDSRNASLPLGAPSPLQPATGDVASDSYNNVFRDTEALRELGVTHYRFSISWARVLPNGSAGVPNREGLRYYRRLLERLRELGVQPVVTLYHWDLPQRLQDAYGGWANRALADHFRDYAELCFRHFGGQVKYWITIDNPYVVAWHGYATGRLAPGIRGSPRLGYLVAHNLLLAHAKVWHLYNTSFRPTQGGQVSIALSSHWINPRRMTDHSIKECQKSLDFVLGWFAKPVFIDGDYPESMKNNLSSILPDFTESEKKFIKGTADFFALCFGPTLSFQLLDPHMKFRQLESPNLRQLLSWIDLEFNHPQIFIVENGWFVSGTTKRDDAKYMYYLKKFIMETLKAIKLDGVDVIGYTAWSLMDGFEWHRGYSIRRGLFYVDFLSQDKMLLPKSSALFYQKLIEKNGFPPLPENQPLEGTFPCDFAWGVVDNYIQVDTTLSQFTDLNVYLWDVHHSKRLIKVDGVVTKKRKSYCVDFAAIQPQIALLQEMHVTHFRFSLDWALILPLGNQSQVNHTILQYYRCMASELVRVNITPVVALWQPMAPNQGLPRLLARQGAWENPYTALAFAEYARLCFQELGHHVKLWITMNEPYTRNMTYSAGHNLLKAHALAWHVYNEKFRHAQNGKISIALQADWIEPACPFSQKDKEVAERVLEFDIGWLAEPIFGSGDYPWVMRDWLNQRNNFLLPYFTEDEKKLIQGTFDFLALSHYTTILVDSEKEDPIKYNDYLEVQEMTDITWLNSPSQVAVVPWGLRKVLNWLKFKYGDLPMYIISNGIDDGLHAEDDQLRVYYMQNYINEALKAHILDGINLCGYFAYSFNDRTAPRFGLYRYAADQFEPKASMKHYRKIIDSNGFPGPETLERFCPEEFTVCTECSFFHTRKSLLAFIAFLFFASIISLSLIFYYSKKGRRSYKL EDYKDDDDK.

Both KLA and KLB have two glycosidase-like domains, one N-terminal andone C-terminal. To identify the region of KLB recognized by the anti-KLBantibodies, hKLB, hKLA and a chimeric construct comprising the hKLAN-terminal glycosidase-like domain and the hKLB c-terminalglycosidase-like domain were cloned into a pCMV-Tag4A mammalianexpression vector (Agilent). The N- and C-terminal domains of hKLA andhKLB correspond to sequences from SEQ ID NO: 151 and SEQ ID NO: 145,respectively, as shown in the Table 5. The N-terminal domains of hKLAand hKLB were divided into 5 segments and the C-terminal domains weredivided into 5 segments based on sequence homology between the twoproteins.

TABLE 5 Subsequence of KLA and KLB. Polypeptide Segment Amino acidsequence N-terminal glycosidase-like 28-469 of SEQ ID NO: 151 domain ofKLA C-terminal glycosidase-like 486-928 of SEQ ID NO: 151 domain of KLAN-terminal glycosidase-like 29-452 of SEQ ID NO: 145 domain of KLBC-terminal glycosidase-like 469-923 of SEQ ID NO: 145 domain of KLBSegment 1 of KLA ECD 1-94 of SEQ ID NO: 151 Segment 2 of KLA ECD 95-201of SEQ ID NO: 151 Segment 3 of KLA ECD 202-329 of SEQ ID NO: 151 Segment4 of KLA ECD 330-442 of SEQ ID NO: 151 Segment 5 of KLA ECD 443-472 ofSEQ ID NO: 151 Segment 6 of KLA ECD 473-529 of SEQ ID NO: 151 Segment 7of KLA ECD 530-613 of SEQ ID NO: 151 Segment 8 of KLA ECD 614-729 of SEQID NO: 151 Segment 9 of KLA ECD 730-831 of SEQ ID NO: 151 Segment 10 ofKLA ECD 832-944 of SEQ ID NO: 151 Segment 1 of KLB ECD 1-77 of SEQ IDNO: 145 Segment 2 of KLB ECD 78-184 of SEQ ID NO: 145 Segment 3 of KLBECD 185-313 of SEQ ID NO: 145 Segment 4 of KLB ECD 314-425 of SEQ ID NO:145 Segment 5 of KLB ECD 426-455 of SEQ ID NO: 145 Segment 6 of KLB ECD456-514 of SEQ ID NO: 145 Segment 7 of KLB ECD 515-598 of SEQ ID NO: 145Segment 8 of KLB ECD 599-722 of SEQ ID NO: 145 Segment 9 of KLB ECD723-829 of SEQ ID NO: 145 Segment 10 of KLB ECD 830-992 of SEQ ID NO:145

A FACS analysis was performed with the antibodies of the presentdisclosure and about half of the antibodies were observed to recognizethe N-terminal glycosidase-like domain of hKLB, whereas others recognizethe C-terminal glycosidase-like domain (Table 6). As shown in Table 6,two of the antibodies that recognized the N-terminal domain of hKLBbound to a portion of the domain comprising segment 1, whereas theothers required only segments 2-5 for binding.

TABLE 6 Mapping of binding of murine anti-KLB antibodies. Anti-KLBAntibody N- or C-terminal domain Segment (1-10) 11F1 N-terminal 1-5 6D12N-terminal 1-5 11D4 N-terminal 2-5 8E1 N-terminal 2-5 46C3 N-terminal2-5 8H7 N-terminal 2-5 21H3 N-terminal 2-5 25F7 N-terminal 2-5 8C5C-terminal 5-10 14E6 C-terminal 5-10 14C6 C-terminal 5-10 24A1C-terminal 5-10 5F8 C-terminal 5-10 6C1 C-terminal 5-10 12A11 C-terminal5-10 12B8 C-terminal 5-10 14C10 C-terminal 5-10

Example 7: Identification of Bispecific Antibodies that SpecificallyActivate the FGFR1c/KLB Complex

Based on the ability of the R1MAbs to activate FGFR1 as a Fab, amolecule that incorporates tethering of a lower affinity R1MAb to ahigher affinity anti-KLB antibody was produced to generate ananti-KLB/anti-FGFR1 bispecific antibody (FIG. 6A; WO2012/158704).

Without being bound to a particular theory, FGF21-mediated activation isproposed to work through the recruitment of FGF21 to the FGFR1c/KLBcomplex through the C-terminal KLB-binding tail, while the determinantsfor FGFR-specificity reside in the N-terminal region, which likely bindsto FGFR via low affinity interaction (See FIG. 6B) (Yie et al. FEBSLett. 583(1): 19-24 (2009)). Therefore, the tethering of anaffinity-lowered R1MAb1 to a high affinity anti-KLB antibody as abispecific antibody could yield a KLB-dependent FGFR1 agonist. Withoutbeing bound to a particular theory, an anti-KLB/anti-FGFR1 bispecificantibody that includes a FGFR1 arm having a low affinity can mitigatethe risk of the anti-KLB/anti-FGFR1 bispecific antibody from binding toFGFR1 tightly in the absence of KLB and preventing the binding and/oractivation of FGFR1 by other FGF ligands (e.g., FGF1, FGF2, FGF8 andFGF23). In addition, an FGFR1 arm with a low affinity can permit thepresence of higher levels of anti-FGFR1 impurities such as, but notlimited to, anti-FGFR1 half-knob antibodies, non-covalent anti-FGFR1dimers, covalent anti-FGFR1 dimers and high-molecular weight species,without resulting in clinically significant side effects.

As used herein, bFKB1, in general, refers to any of the severalanti-KLB/anti-FGFR1 bispecific antibodies disclosed herein. Detailsregarding the specific anti-KLB/anti-FGFR1 bispecific antibodiesdisclosed in the Figures are described below. HEK293 cells wereco-transfected with a mixture of four expression vectors encoding theheavy and light chains of anti-FGFR1 (YW182.2 (R1MAb1) and YW182.3(R1MAb2)) and the anti-KLB antibodies described above. The heavy chainof anti-FGFR1 and anti-KLB were respectively tagged with the Flagpeptide and Oct-Histidine so that heterodimeric IgG could be purified bysequential affinity purification from conditioned medium. Partiallypurified heterodimeric IgG were then analyzed in a GAL-ELK1 basedluciferase assay to identify KLB-dependent agonists. To minimizemispairing of heavy and light chains, anti-FGFR1 was expressed withhuman Fab constant region, and anti-KLB was expressed with mouse Fabconstant region. The tagged-bispecific IgGs were initially tested in acrude form using 28 combinations of 3 anti-R1MAbs and 18 anti-KLB Abs(Table 7).

FIG. 7A shows induction data for certain bispecific combinations ofYW182.2 (R1MAb1), YW182.3 (R1MAb2) and YW182.5 (R1MAb3) with 18C5, 12A11and 14E6 in a GAL-ELK1 luciferase assay. In most cases, it was observedthat the bispecific antibodies activated signaling significantly betterin cells that coexpressed FGFR1c and KLB compared with cells thatexpressed only FGFR1c, but not KLB.

Based on the activity of these antibodies in these initial experiments,8 representative anti-KLB Abs (Ph #5, 8C5, 12A11, 14C10, 6D12, 11D4, 6C1and, as a negative control, 14E6) were used to produce un-taggedbispecific antibodies with YW182.5 (by using a previously describedknob-hole technology for further characterization (supra, and, e.g.,Atwell, et al. FEBS Lett. 583(1): 19-24 (2009)). As shown in FIG. 8A,bispecific antibodies were produced with human IgG1 constant region(wild-type, with effector function (1)) and with human IgG1 constantregion with N297G mutation to eliminate the effector function (3), ormouse constant region with dual [D265G/N297G] mutations (DANG) toeliminate effector function (2).

Table 7 below lists various bispecific antibodies that were made usingthe knob-in-hole technology.

TABLE 7 Bispecific anti-KLB/anti-FGFR1 antibodies. BsAb Anti-FGFR1Anti-FGFR1 Anti-KLB ID# Arm Platform Anti-KLB Arm Platform 1 YW182.3Human IgG1 Ph#5 Human IgG1 2 YW182.2 Human IgG1 Ph#5 Human IgG1 3YW182.3 Human IgG1 14E6 Murine VH/VL- Human IgG1 chimera 4 YW182.3 HumanIgG1 8C5 (KLBmAb1) Murine VH/VL- Human IgG1 chimera 5 YW182.5 Human IgG111D4 (KLBmAb5) Murine VH/VL- Human IgG1 chimera 6 YW182.5 Human IgG114C10 (KLBmAb3) Murine VH/VL- Human IgG1 chimera 7 YW182.5 Human IgG16C1 (KLBmAb4) Murine VH/VL- Human IgG1 chimera 8 YW182.5 Human IgG1 6D12(KLBmAb6) Murine VH/VL- Human IgG1 chimera 9 YW182.5 Human IgG1 12A11(KLBmAb2) Murine VH/VL- Human IgG1 chimera 10 YW182.5 Human IgG1 8C5(KLBmAb1) Murine VH/VL- Human IgG1 chimera 11 YW182.5 Human IgG18C5.K4H3.RNL Human IgG1 N297G N297G 12 YW182.5 Human IgG1 8C5.K4H3.KNVHuman IgG1 N297G N297G 13 YW182.5 Human IgG1 8C5.K4H3.M4L.KNV Human IgG1N297G 14 YW182.5 Human IgG1 8C5.K4H3.M4L.KNV Human IgG1 N297G N297G 15YW182.5_W33Y Human IgG1 8C5.K4H3.M4L.KNV Human IgG1 N297G N297G 16YW182.2_W33Y Human IgG1 8C5.K4H3.M4L.KNV Human IgG1 N297G N297G 17YW182.5_YGDY Human IgG1 8C5.K4H3.M4L.KNV Human IgG1 N297G N297G 18YW182.2_YA Human IgG1 8C5.K4H3.M4L.KNV Human IgG1 N297G N297G 19 YW182.5Human IgG1 8C5_W52Y.K4H3.M4L.KNV Human IgG1 N297G N297G 20 YW182.5 HumanVH/VL- Murine 8C5 Murine IgG2a Murine IgG2a DANG chimera DANG

The isotype control IgG used was either anti-ragweed (murine IgG2a) orthe anti-human Her2 trastuzumab (human IgG1). Fab fragments wereexpressed in E. coli and purified using conventional columnchromatography. Recombinant FGF21 was from R&D systems (2539-FG/CF)except for radioligand cell binding assay, which was performed withiodinated FGF21 from Phoenix Pharmaceuticals and in-house producedunlabeled FGF21. Each of the bispecific combinations (except for thenegative control) showed signaling dependent on both FGFR1c and KLB. Thedata for certain combinations are shown in FIG. 7B. In addition, thecombination of the anti-KLB arms with the YW182.5 (R1MAb3) arm showedlower background signaling in cells that expressed FGFR1c, but not KLB.

As shown in FIG. 6C, the activity of an anti-KLB/anti-FGFR1c antibody(BsAb17) was tested in FGFR1-deficient HEK293 cells expressing variousreceptors. FGFR1-deficient HEK293T cells were generated using theCRISPR-cas9 method using guide RNAs. The anti-KLB/anti-FGFR1c antibodywas observed to induce luciferase activity in cells coexpressingrecombinant hFGFR1c and hKLB (FIG. 6C).

Similar results were observed for other anti-KLB/anti-FGFR1c antibodies.As shown in FIG. 7C, when tested in a GAL-ELK1-based luciferase assay inHEK293 cells expressing FGFR1c with or without KLB, multiple bispecificantibody combinations of anti-FGFR1 and anti-KLB arms, e.g., BsAb5, 6,7, 8, 9, 10, induced luciferase activity in a dose-dependent manner incells expressing recombinant hFGFR1c and hKLB, but not in cells withoutKLB expression. These results indicate that these bispecific antibodiesact as KLB-dependent FGFR agonists, just like FGF21.

Synergy of an anti-KLB/anti-FGFR1c antibody (BsAb17) with FGF21 was alsotested. As shown in FIG. 9B, no synergy between BsAb17 and FGF21 wasobserved when the concentration of FGF21 was increased incrementally andthe concentration of BsAb17 remained unchanged.

In addition, as the concentration of the anti-KLB/anti-FGFR1c antibody(BsAb17) was increased incrementally and the concentration of FGF21remained unchanged no synergy between BsAb17 and FGF21 was observed(FIG. 9C).

The solution binding affinity (K_(d)) of two of the bispecificantibodies, BsAb10 and BsAb9, (along with hFGF21) to HEK293 cellsexpressing KLB from human, cynomolgous monkey and mice, human FGFR1c, orboth hFGFR1c and hKLB was measured by a radiolabeled ligand bindingassay. For the radioligand cell binding assay, HEK293 cells that stablyco-expressing KLB and/or FGFR1c were placed into 96-well plate at adensity of 100,000 to 200,000 cells per 0.2 mL in binding buffer (DMEMwith 1% bovine serum albumin (BSA), 50 mM HEPES, pH 7.2, 0.1% sodiumazide and 350 mM human IgG). Competition reaction mixtures of 50 μLcontaining a fixed concentration of iodinated FGF21 (PhoenixPharmaceuticals) or iodinated BsAb, and serially diluted concentrationsof unlabeled FGF21 (Genentech) or unlabeled BsAb were added to thecells. Competition reactions with cells were incubated for 2 h at roomtemperature. After the 2 h incubation, the competition reactions weretransferred to a Millipore Multiscreen filter plate and washed fourtimes with binding buffer to separate the free from bound iodinatedFGF21 or antibody. The filters were counted on a Wallac Wizard 1470gamma counter (PerkinElmer Life and Analytical Sciences). The bindingdata were evaluated using New Ligand software (Genentech), which usesthe fitting algorithm of Munson and Rodbard (Munson and Rodbard Anal.Biochem. 107, 220-239 (1980)) to determine the binding affinity.

As shown in Table 8, both antibodies exhibited some good reactivity tocells expressing only KLB (in a cross-species pattern consistent withthat observed previously), but both bound much more weakly to cellsexpressing only hFGFR1c and more strongly to cells expressing both hKLBand hFGFR1c.

TABLE 8 Binding of bispecific anti-KLB antibodies to KLB/FGFR1 fromdifferent species. FGFR1c (none) (none) (none) Human Human KLB HumanCyno Mouse Human (none) BsAb10 6.6 nM 15.4 nM 15.5 nM 2.3 nM 300 nMBsAb9 9.8 nM   35 nM n.d. 2.2 nM 300 nM hFGF21 n.d. n.d. n.d. 5.3 nMn.d.

FIG. 9D shows the affinity of BsAb10 and BsAb9 to HEK293 cells stablyexpressing hKLB, hFGFR1c, or both, as compared to an antibody with twocorresponding anti-FGFR1-binding arms (YW182.5) using FACS analysis.Similar results were obtained to those indicated above (FIG. 9D).

Further experiments were conducted with one bispecific antibody, BsAb10,which has YW182.5 as the anti-FGFR1 arm and 8C5 as the anti-KLB arm, andderivatives of BsAb10 (BsAb11-20). As shown in FIG. 6C, murine receptorswere expressed in HEK293 cells and showed that BsAb17 induced luciferaseactivity in these cells as well, confirming the species cross reactivityof this Ab.

BsAb10 was next tested in rat L6 myoblast cells lacking endogenous KLBand FGFRs, but transfected to express hKLB and each of 5 hFGFR isoforms(FIG. 9A). BsAb10 was found to induce luciferase activity only in cellsexpressing both FGFR1c and KLB, indicating that BsAb10 acts as aspecific agonist for the FGFR1c/KLB complex but not KLB in complex withother FGFRs (FIG. 9A). FGF21 and FGF19 were used as controls todemonstrate that FGF21 induced luciferase activity when cells expresseda combination of KLB and any one of FGFR1c, 2c, or 3c, and FGF19 inducedactivity in cells that expressed a combination of KLB and FGFR4.Recombinant FGF21 was from R&D systems (2539-FG/CF) except forradioligand cell binding assay, which was performed with iodinated FGF21from Phoenix Pharmaceuticals and in-house produced unlabeled FGF21.cDNAs encoding the extracellular domain (ECD) of human FGFR1b, 1c, 2b,2c, 3b, 3c, and 4 were cloned into expression vector containing thecytomegalovirus (CMV) promoter to generate human FGFR-human Fc chimericproteins or His-tagged FGFR proteins.

However, as described above, the parental anti-FGFR1c antibody, R1MAb3(YW182.5) of BsAb10 can, surprisingly, binds to FGFR1b, an isoform ofFGFR1 that does not interact with KLB. In addition, R1MAb3 (YW182.5) andcan activate FGFR1b in the GAL-ELK1 assay in L6 cells, which is incontrast to the activity of BsAb10 (see FIGS. 2C and 2B).

Further, a combination of FGFR1b and KLB did not support activation byBsAb10 (FIG. 9A). Without being bound to a particular theory, these datasuggest that the presence of preformed FGFR1/KLB complex is aprerequisite for the KLB-dependent activation of FGFR1 by BsAb10.

Example 8: BsAb10, and its Derivatives, Act as Molecular Mimetics ofFGF21

Further characterizations of BsAb10 and its derivatives (BsAb11-20) andFGF21 revealed some similarities and differences. To determine thephosphorylation level of the MAPK signaling intermediates, cells weregrown in preadipocyte basal medium-2 containing FBS, L-glutamine andGA-1000. Once confluent, subcutaneous pre-adipocytes (acquired fromLonza) were differentiated in growth media containing dexamethasone,indomethacin, and 3-isobutyl-1-methylxanthine (IBMX). For geneexpression analysis, cells were differentiated for 14 days, and thenfurther cultured for additional 48 h with indicated agonists. For MAPKsignaling analysis, cells were differentiated for 10 days, grown inserum-free medium for 3 h, and then further cultured for an additional hwith indicated agonists.

As shown in FIG. 6D, BsAb10, BsAb17, BsAb20, and FGF21 showed acomparable activity to induce phosphorylation of the MAPK signalingintermediates such as MEK and ERK in primary human adipocytes, whichrepresent the relevant cell type for the anti-diabetic activity ofFGF21, as determined by western blot. Antibodies used for the Westernblot analysis were from Cell Signaling Technology: pFRS2a (T196)(#3864), pMEK1/2 (S217/221) (#9154), pERK1/2 (T202/204)(#4370), ERK1/2(#4695), HSP90 (#4874), j-Actin (#5125), from abeam: UCP1 (ab10983), orfrom R&D Systems: KLB (AF2619).

As shown in FIG. 8B, an increase in the phosphorylation of ERK,represented as a fold change in pERK levels, was observed in primaryhuman adipocytes treated with BsAb10, BsAb17, BsAb20 or FGF21.

In addition, the affinity profile of BsAb10 resembles that of FGF21.When tested by FACS, BsAb10 showed strong binding to cells expressinghKLB, whether or not FGFR1c was coexpressed (FIG. 10A). Somewhatsurprisingly, very little binding of BsAb10 was observed when cellsexpressed FGFR1c, but not KLB, indicating that monovalent affinity ofthe YW182.5 arm is extremely low (FIG. 10A).

As shown in FIG. 10B, a radiolabelled-ligand assay indicated that thedissociation constant (K_(d)) of BsAb10 to the cells expressing bothFGFR1c and KLB is 2.3 nM, close to 5.3 nM observed for hFGF21 in asimilar assay format. These values were close to the observed EC₅₀ ofthese molecules in GAL-ELK1 assay in HEK293 cells (3.2 nM and 4.7 nM,respectively for BsAb10 and FGF21. When cells expressing human KLBalone, or mouse KLB alone, the K_(d) were 6.6 nM and 15.5 nM,respectively.

Since the affinity to FGFR1 was so low, the radiolabel ligand assaycould not reliably determine the K_(d) of BsAb10 to the cells expressingonly FGFR1c, but it was estimated to be >300 nM, as shown in FIG. 3B.Due to a similar reason, binding kinetics of BsAb10 to FGFR1 could notbe reliably determined by SPR either.

Further, the interaction between FGFR1c-ECD and KLB-ECD proteins werestablized by BsAb10 as previously observed for FGF21, consistent withthe notion that BsAb10 acts as a FGFR1c-selective FGF-21 mimetic (FIG.11 ) (Yie et al., Chemical Biology; Drug Design 79, 398-410 (2012)).FGFR1/KLB/BsAb10 interaction was studied by surface plasmon resonance(SPR) measurements on a PROTEON™ XPR36 (Bio-Rad Laboratories) instrumentat 25° C. FGFR1-HIS protein (20 μg/ml) at pH4.5 was immobilized atsurface density (1000 RU) on an activated PROTEON™ GLC sensor chip usingstandard amine coupling procedures as described by the manufacturer.BsAb10 and/or 1:1 mixtures of BsAb10 and KLB-ECD were injected at 6.25nM, 12.5 nM, 25 nM, 50 nM, 100 nM, or 200 nM in PBS containing 0.005%v/v TWEEN®-20, 0.3M NaCl (pH7.4) at a flow rate of 80 μl/min andsensorgrams for association and disassociation phases were recorded.Analytes were injected for 300 sec and allowed to disassociate for 600sec. Data was referenced with interspots, processed, and disassociationconstants measured with the PROTEON™ Manager software (version 3.0,Bio-Rad). The activation of FGFR1c/KLB complex by BsAb10 suggested aternary complex formation by FGFR1c-ECD, KLB-ECD and BsAb10.

As shown in FIG. 11 , it was also observed that BsAb10 formed a ternarycomplex with recombinant KLB-ECD and FGF21 or FGF19. BsAb10/KLB/FGFinteraction was studied by bio-layer interferometry (BLI) measurementson an Octet RED (ForteBio) instrument at 25° C. BsAb10 (20 μg/ml) at pH4.5 was immobilized on activated amine reactive biosensor tips asdescribed by the manufacturer. KLB-ECD (20 μg/ml) in PBS containing0.005% v/v TWEEN®-20, 0.3M NaCl (pH 7.4) was captured onto the samebiosensor tips and measured with FGF21 (R&D Systems) at 0, 0.2, 0.8, or2 μM in the same buffer. Qualitative data was processed with the dataacquisition software (ForteBio).

FIG. 12A shows a schematic of a cell-surface time-resolved fluorescenceresonance energy transfer (TR-FRET) experiment that was performed. ForTR-FRET, COS7 cells were co-transfected to express SNAP-tagged FGFR1 anduntagged KLB and seeded in a white bottom 96-well plate (Costar) at100,000 cells per well. Transfected cells were labeled 24 hpost-transfection with 100 nM of donor-conjugated benzylguanineSNAP-Lumi4-Tb (Cisbio) and 1 μM of acceptor-conjugated benzyl-guanineSNAPAlexa647 (NEB) for 1 h at 37° C., 5% CO₂. After three washes, theLumi4-Tb emission and the TR-FRET signal were recorded at 620 nm and 665nm, respectively, for 400 s after a 60 s delay following laserexcitation at 343 nm using a Safire2 plate reader (Tecan) at t=0 andt=15 min after ligand addition. The emission signal of the Alexa647 wasdetected at 682 nm after excitation at 640 nm using the same platereader. FRET intensity was then calculated as: (signal at 665 nm fromcells labeled with SNAP-donor and SNAP-acceptor)−(signal at 665 nm fromthe same batch of transfected cells labeled with SNAP-donor and nonlabeled SNAP).

As shown in FIG. 12B, the TR-FRET experiment suggested that both BsAb17and FGF21 enhances dimerization of FGFR1c-ECD when KLB is also presentin the cell. The results were shown as FRET ratio: FRET intensitydivided by the donor emission at 620 nm.

In addition, BsAb10 binds to the C-terminal half of KLB-ECD, whereasFGF21 and FGF19 have been thought to bind to the same site on KLB in theN-terminal half (Goetz et al. Mol. Cell. Biol. 32(10): 1944-54 (2012);Foltz et al. Sci. Transl. Med. 4: 162ra153 (2012)), which suggests thatthe epitope of BsAb10 on KLB should be distinct from the FGF21 and FGF19binding site. In order to map the KLB epitope for BsAb10, binding of 8C5(the KLB-binding arm of BsAb10) to a series of chimeric antigensexpressed in HEK293 cells. Each chimera was constructed by fusing humanKLB and human Klotho alpha (KLA) protein (50% identity to human KLBproteins) or rabbit KLB (86% identity to human KLB). As summarized inFIG. 13A, 8C5 binds the C-terminal domain of KLB, in particular, in theregion containing 34 amino acids in the C-terminal domain of KLB(SSPTRLAVIPWGVRKLLRWVRRNYGDMIDIYITAS; SEQ ID NO: 142).

As shown in FIG. 13B, the amino acid sequence of SEQ ID NO: 142 cancorrespond to amino acids 857-890 of a KLB protein that includes asignal sequence, e.g., such as a 52 amino acid sequence having thesequence set forth in SEQ ID NO: 157, or can refer to amino acids805-838 of a KLB protein that does not include a signal sequence.

Despite the similarity between FGF21 and BsAb10 and its derivatives inthe downstream action, the epitope of BsAb10 on KLB is distinct from theFGF21 and FGF19 binding site (FIG. 14A).

FIG. 14B shows the results of a GAL-ELK1 luciferase assay performed inrat L6 myoblast cells co-transfected with FGFR4 and KLB and treated withFGF19 alone or in combination with an anti-KLB/anti-FGFR1c antibody(BsAb17). As shown in FIG. 14B, BsAb17 pretreatment also did not blockFGF19-activity in L6 cells expressing FGFR4/KLB complex.

Additionally, and as shown in FIG. 14C, BsAb17 pretreatment did notblock FGF19-activity in H4IIE hepatoma cells expressing FGFR4 and KLB.In the presence of BsAb17, FGF19 was still able to activate theFGFR4/KLB complex to induce phosphorylation of ERK (FIG. 14C). Thesedata indicate that the disclosed anti-KLB/anti-FGFR1 bispecificantibody, e.g., an anti-KLB/anti-FGFR1c bispecific antibody, does notinterfere with the interaction of FGF19 or FGF21 with the KLB/FGFR1ccomplex.

Example 9: BsAb10, and its Derivatives, Act as a Long Acting FGF21Mimetic In Vivo

The cross-reactivity of BsAb10 and its derivatives with murine receptorcomplex as described above (see, e.g., FIGS. 6C and 10B) allowed thetesting of its in vivo activity in mouse models. To avoid potentialtoxicity from the IgG effector function, a dual mutation [D265A/N297G]was introduced to BsAb20 in the Fc region that abolishes binding toFcgRs and recruitment of immune effector cells. In addition, to avoidpotential toxicity from the IgG effector function, N297G was introducedto BsAb17 in the Fc region that abolishes binding to FcgRs andrecruitment of immune effector cells.

As shown in FIG. 15A, when i.p. injected into diabetic db/db mice at 5mg/kg, BsAb17 reduced blood glucose levels to a similar extent withoutaffecting food intake or body weight. Lean C57BL/6 mice treated withBsAb17 showed reduced blood glucose, but did not achieve toxichypoglycemia (FIG. 15A).

In addition, when high fat diet-fed C57BL/6 mice (Diet Induced Obesity,DIO) were injected with BsAb17 at 3 mg/kg on day 0 and 6, significantreductions in weight loss and blood glucose were observed (FIG. 15B).For high-fat diet feeding, a high fat, high carbohydrate diet (HarlanTeklad TD.03584, 58.4% calories from fat) was used.

As shown in FIG. 15C, an improvement in glucose tolerance was observedin high fat diet-fed C57BL/6 mice (Diet Induced Obesity, DIO) that wereinjected with BsAb17 at 3 mg/kg.

Reductions in hepatic triglyceride, serum insulin, free fatty acid,triglyceride and total cholesterol were also observed in high fatdiet-fed C57BL/6 mice (Diet Induced Obesity, DIO) that were injectedwith BsAb17 at 3 mg/kg (FIG. 15D). Similar results were previouslyobserved with FGF21 injections.

A separate experiment was performed in klb heterozygous mice andhomozygous klb deficient mice to determine if the improvement in glucosetolerance observed upon treatment with an anti-KLB/anti-FGFR1cbispecific antibody requires functional KLB. To generate klb-deficient(KO) mice, a Klb-specific Zinc Finger Nuclease (ZFN) pair was obtainedfrom Sigma-Aldrich and used for pronuclear microinjection according toestablished methods. The ZFN pair targets the following Klb sequence inthe mouse genome (cut site in small letters), and the KO mice lack onebp deletion (g in bold) causing a frameshift:GTTACCGGCTTCtccggaGACGGGAAAGCAATATGG (SEQ ID NO: 156). FIG. 16A showsthe N-terminal amino acid sequence of mouse KLB protein and thecorresponding amino acid sequence encoded by the klb allele in the klbdeficient mice.

FIG. 16B shows the results of a western blot that was performed toconfirm the lack of KLB protein expression in klb deficient mice.

As shown in FIG. 16C, BsAb20 improved the glucose tolerance in klbheterozygous mice as measured by the glucose tolerance test (GTT), butnot homozygous klb deficient mice, indicating that the improvements inglucose tolerance require functional KLB. For the glucose tolerance test(GTT), mice were fasted overnight and i.p. injected with 2 g/kg glucosesolution.

In addition, unlike anti-FGFR1 R1MAb1, which alters the levels of serumFGF23 and phosphorus (Wu et al., Sci Transl Med 3, 113ra126 (2011) andWu et al., PLoS One 8, e57322 (2013)), the anti-KLB/anti-FGFR1bispecific antibody did not affect these serum parameters, indicatingthe absence of KLB-independent FGFR1 agonistic activity (FIG. 16D).

As shown in FIG. 17 , BsAb17 did not alter serum FGF23 or phosphorouslevels, which are sensitive markers of KLB-independent FGFR1. Insulinaction in BsAb17 treated mice was measured byhyperinsulinemic-euglycemic clamp. In brief, mice were anesthetized withisoflurane and the left common carotid artery and right jugular veinwere catheterized for sampling and infusing, respectively. The free endsof the catheters were tunneled under the skin to the back of the neckwhere the loose ends of the catheters were attached to tubing made ofMICRO-RENATHANE® (0.033 in OD). Animals were individually housed aftersurgery and body weight was recorded daily. All metabolic experimentswere performed following a 5-day postoperative recovery period and havebeen previously described. Conscious, unrestrained mice were placed in a1-L plastic container lined with bedding and fasted at 7:00 am (t=−300min). The mice were immediately connected to a Dual Channel StainlessSteel Swivel (Instech Laboratories) to allow simultaneous jugular veininfusion and sampling of arterial blood. Mice were not handled and wereallowed to move freely to eliminate stress. 2 h prior to initiation ofthe clamp a 5 μCi bolus of [3-3H]-D-glucose was given into the jugularvein (t=−120 min) this followed by a constant infusion at a rate at 0.05μCi/min. Following a 2 h equilibrium period at t=0 min (i.e., a 5 hfast) a baseline arterial blood sample was drawn for measurement ofblood glucose, [3-3H]-D-glucose, hematocrit and plasma insulin. A 145min hyperinsulinemic-euglycemic (4 mU/kg/min) clamp was then initiated.[3-3H]-D-glucose was added to the variable glucose infusion that wasused to maintain euglycemia and the constant infusion of[3-3H]-D-glucose was discontinued so as to clamp arterial glucosespecific activity at a constant level. Red blood cells from a donormouse on a C57Bl/6J background were washed with and reconstituted in anequal volume of 0.9% heparinized saline (hematocrit ˜50%) and infused ata rate of 4 μl/min for the duration of the study to replace bloodremoved during study. Arterial blood samples were taken every tenminutes to determine blood glucose levels. At t=80, 90, 100 and 120 min,blood samples were taken to determine [3-3H]-D-glucose. At t=120 min, a13 μCi bolus of 2-deoxy [14C] glucose ([2-14C]DG) was administered intothe jugular vein catheter. At t=122, 125, 130, 135, and 145 min arterialblood was sampled to determine blood glucose, plasma [3-3H]-D-glucoseand [2-14C]DG. Arterial insulin concentration was measured at 100 and120 min. At t=145 min mice were then anesthetized. The soleus,gastrocnemius, white superficial vastus lateralis (Quad), liver, heart,epididymal and subcutaneous white adipose tissue, brown adipose tissueand brain were excised, immediately frozen in liquid nitrogen, andstored at −70° C. until future tissue analysis. Immunoreactive insulinwas assayed using a Linco Rat Radioimmunoassay kit (LincoResearch).

To measure [3⁻³H]-D-glucose, plasma samples were deproteinized withbarium hydroxide (Ba(OH)₂) and zinc sulfate (ZnSO₄), dried, andradioactivity was determined using liquid scintillation counting.Excised tissues were deproteinized with perchloric acid and thenneutralized to a pH of ˜7.5. A portion of the sample was counted([2-¹⁴C]DG and [2-¹⁴C]DG-Gphosphate ([2-¹⁴C]DGP) and a portion wastreated with Ba(OH)₂ and ZnSO₄ and the supernatant was counted([2-¹⁴C]DG). Both [2-¹⁴C]DG and [2-¹⁴C]DG-phosphate ([2-¹⁴C]DGP)radioactivity levels were determined using liquid scintillationcounting. Glucose flux rates were assessed using non-steady stateequations assuming a volume of distribution (130 ml/kg). Tissue-specificclearance (K_(g)) of [2-¹⁴C]DG and an index of glucose uptake (R_(g))was calculated as previously described (Kraegen, E. W. et al., Am. J.Physiol. 248, E353-362 (1985)): K_(g)=[2-¹⁴C]DGP_(tissue)/AUC[2-¹⁴C]DG_(plasma), R_(g)=K_(g)×[glucose]_(plasma), where[2-¹⁴C]DGP_(tissue) is the [2-¹⁴C]DGP radioactivity (dpm/g) in thetissue, AUC [2-¹⁴C]DG_(plasma) is the area under the plasma [2-¹⁴C]DGdisappearance curve (dpm/mL/min), and [glucose]_(plasma) is the averageblood glucose (g/l) during the experimental period (t=102-125 min). Dataare presented as mean±SEM.

As shown in FIG. 15E, which depicts whole body glucose utilizationmeasured following a single injection of BsAb17 at 10 mg/kg, BsAb17improved the rates of insulin stimulated whole body glucose utilization.

In addition, and as shown in FIG. 15F, BsAb17 improved insulinsuppression of endogenous glucose production rates following a singleinjection of BsAb17 at 10 mg/kg. These results indicate that a singleinjection of 10 mg/kg of BsAb17 in DIO mice 5 days prior to the clampmarkedly lowered fasted glucose and insulin concentrations.

Tissue glucose uptake (R_(g)) at the end of the insulin-stimulatedperiod was enhanced in heart, skeletal muscle, white adipose tissues(WAT) and interscapular BAT tissue (iBAT), indicating whole body insulinsensitization by BsAb17 (FIG. 15G).

The amount of arterial blood glucose excursion was determined during theclamp experiment. As shown in FIG. 18A, the amount of arterial bloodglucose excursion was different between mice injected with BsAb17 versusmice injected with control IgG during the hyperinsulinemic-euglycemicclamp experiment.

The difference in weight between mice injected with BsAb17 versus miceinjected with control IgG was also determined. As shown in FIG. 18B, thechanges observed in glucose and insulin concentrations were without anapparent loss in weight.

The steady state glucose infusion rate was also analyzed following aninjection with BsAb17. As shown in FIG. 18C, the steady state glucoseinfusion rate was increased by 64% following a BsAb17 injection. Theseresults demonstrate that BsAb17 improved whole body insulin sensitivityin DIO mice even before weight loss becomes apparent.

Previous studies with pharmacological doses of FGF19 or FGF21 have shownincreased energy expenditure (EE) (Fu et al., Endocrinology 145,2594-2603 (2004); Coskun et al., Endocrinology 149, 6018-6027 (2008); Wuet al., PLoS One 8, e57322 (2013); Lin et al., Cell Metab 17, 779-789(2013)), thus it was reasoned that a similar effect would be observed.The following equations were used to calculate EE and Respiratoryquotient (RQ). EE=VO₂X(3.815+1.232×RQ), where (RQ=VCO₂/VO₂). Indeed,single BsAb17 injection into DIO or lean mice at normal room temperature(21° C.) led to significant increase in 02 consumption (VO₂), CO₂production (VCO₂), and EE per injected animal without significant changein activity count (FIG. 19A). Unexpectedly, the observed 15-46% increasein EE did not accompany significant changes in respiratory quotients(RQ=VCO₂/VO₂) (FIG. 19A).

A similar increase in EE without change in RQ was elicited bycontinuously infusing FGF21 into DIO mice (FIG. 21C).

FIG. 20A shows the amount of VO₂, VCO₂ and total activity counts of DIOmice treated with a single BsAb17 injection at normal room temperature.

As shown in FIG. 19B, the increase in EE was sustained when the cagetemperature was elevated to thermoneutrality (29-30° C.), suggestingthat BsAb17-induction of brown fat activation does not rely on adaptivethermogenic input from the sympathetic nervous system.

FIG. 20B shows the amount of VO₂, VCO₂ and total activity counts of DIOmice treated with a single BsAb17 injection at normal room temperaturefollowed by a shift in temperature to thermoneutrality.

An increase in EE was also evident when DIO mice acclimated atthermoneutral room temperature (29-30° C.) were tested for two weeks(FIG. 21B).

As summarized in FIG. 21A, which shows the average EE values, changes inEE were observed in lean and DIO mice at normal room temperature and inlean and DIO mice acclimated at thermoneutral room temperature.

In contrast, a continuous infusion with β3-specific adrenoceptor agonistCL-316,243 induced an acute increase in EE and reduction in RQ asanticipated (FIG. 19H). Continuous infusion of FGF21 or CL-316,243 wasperformed using an osmotic mini-pump (Alzet 2001) that wassubcutaneously implanted. Thus, the BsAb17- and FGF21-induced EE isrobust, but appears more selective than other previously described BATactivation mechanisms, such as administration of sympathomimetics(norepinephrine or β3-specific adrenoceptor agonist CL-316,243), cardiacnatriuretic peptides, or Interleukin-4, that accompany promotion oflipid oxidation and reduction in RQ (Gerhart-Hines et al., Mol. Cell 44,851-863 (2011); Mattsson et al., American journal of physiology.Endocrinology and metabolism 299, E374-383 (2010); Nguyen et al., Nature480, 104-108 (2011); Birkenfeld et al. Diabetes 57, 3199-3204 (2008);Bordicchia et al., J. Clin. Invest. 122, 1022-1036 (2012); and de Souzaet al., Diabetes 46, 1257-1263 (1997)).

Without being bound to a particular theory, several lines of evidencesuggested the dominant role of BAT activation in the metabolic action ofBsAb17. First, as shown in FIG. 19C, BsAb17 injection increased uptakeof 18F-Fludeoxyglucose (FDG) specifically into iBAT.

Second, a single BsAb17 injection induced UCP1 protein expression ininguinal WAT (ingWAT), which is indicative of adipose tissue browning(FIG. 19D).

As shown in FIG. 19E, induction of UCP1 expression was also observed incultured primary adipocytes treated with FGF21 or BsAb17, indicatingdirect action on mature adipocytes. To determine UCP1 expression, totalRNA was used to synthesize cDNA using SUPERSCRIPT® VILO cDNA SynthesisKit (ABI). For qPCR, samples were run in triplicate in the ViiA 7Real-Time PCR instrument (Applied Biosystems). The Applied Biosystemspredesigned TAQMAN® Gene Expression Assay probe used was UCP1(Hs01027785_m1). For each sample, mRNA abundance was normalized to theamount of TBP (Hs00427620_m1) and SDHA (Hs00188166_m1) transcripts.

Third, using telemetry system, an increase in resting core bodytemperature was observed after single BsAb17 injection that lasted for≥26 days before gradually returning to baseline (FIG. 19F).

FIG. 22 shows the differences in the core body temperature that wasobserved in mice after a single BsAb17 injection compared to micetreated with control IgG. Core body temperatures were monitored using aTA-F10 transmitter (Data Sciences International, DSI) that wassurgically implanted into peritoneal cavity. After recovery from thesurgery, mice were randomized into groups based on body weight and corebody temperature. Core body temperature and activity were monitoredusing DSI Implantable Telemetry System.

Gene expression profiles were analyzed in iBAT of DIO mice that receiveda single injection of BsAb17, FGF21 or control IgG. As shown in FIG.19G, single BsAb17 injection induced gene expression changes in iBATthat resembles twice-daily injections with FGF21.

Finally, when injected into C57BL/6 mice, both FGF21 and BsAb20 inducedERK and MEK phosphorylation in various adipose tissues, including iBATand ingWAT (FIG. 23 ).

In previous studies, adiponectin was suggested to contribute to the fullaction of FGF21 (Lin et al., Cell Metab 17, 779-789 (2013); Holland etal., Cell Metab 17, 790-797 (2013)). Indeed, single injection of BsAb17into DIO mice led to an increase in serum high molecular weight (HMW)adiponectin levels, with associated weight loss (FIG. 24A).

Similarly, a single injection of BsAb17 into lean cynomolgus monkeys(FIG. 24B) led to an increase in serum high molecular weight (HMW)adiponectin levels, with associated weight loss.

As shown in FIG. 24C, upon single injection of BsAb17, adiponectin(Adipoq) KO mice on HFD exhibited a robust response in elevating EE(25.3% increase vs 20.9% increase in wt mice).

In addition, upon single injection of BsAb17, adiponectin (Adipoq) KOmice on HFD exhibited reduced body weight and hepatic triglyceridelevels (FIG. 24D). However, the response in glucose tolerance, insulintolerance, changes in serum insulin and various lipids were all somewhatblunted in the KO mice (FIG. 24D), consistent with the idea that BsAb17acts as a FGF21 mimetic in regulating whole body nutrient metabolism inpart via adiponectin function. For determining insulin tolerance, micewere fasted for 4 h, and i.p. injected with 1 U/kg human insulinsolution (Humulin R, Eli Lilly and Company).

The heightened receptor selectivity of BsAb10 (see FIG. 9A) andpreviously described low brain penetrance of IgG molecules (Yu et al.,Sci Transl Med 3, 84ra44 (2011)) predict an altered safety profile ofBsAb10 and its derivatives compared with FGF21/19. Consistent with thelow expression level of FGFR1 in the liver, FGF21, but not BsAb17,induced mRNA expression of classical FGFR target genes Spry4 and Dusp6in the liver (FIG. 25 ).

BsAb17 or BsAb20 also resulted in an increased phospho-ERK signal invarious adipose tissues and pancreatic acinar cells, but not in theliver (FIG. 23 ).

FIG. 26 shows that BsAb17 also does not increase the phosphorylation ofERK in various brain sections including circumventricular organs, asdetermined by immunohistochemistry.

In addition, chronic BsAb20 treatment of DIO mice for 8 weeks reducedthe number of BrdU+ cells in the liver to the level of lean C57BL/6mice, the opposite of what was expected for FGF19-like activity (FIG. 27). For hepatic BrdU incorporation, mice were intraperitoneally injectedwith 100 mg/kg BrdU (BD Biosciences) at 2 h prior to euthanasia.Anti-BrdU staining was carried out as described (Nicholes, K., et al.Am. J. Pathol. 160, 2295-2307 (2002)) and BrdU positive hepatocytes werecounted by using the Ariol automated image analysis system.

Bone analysis of mice that were treated with an anti-KLB/anti-FGFR1cantibody was performed. FIG. 28A shows a schematic representation of theanalysis. To perform the bone analysis, femur samples were imaged by aSCANCO Medical (Basserdorf, Switzerland) CT40 micro-imaging systemoperating with x-ray tube energy level a 70 keV and a current of 114microamperes. Contiguous axial image slices were obtained with anisotropic voxel size of 12 m. Morphometric analysis of the trabecularbone within the femur was performed with the SCANCO Medical (Basserdorf,Switzerland) CT40 evaluation software. Semi-automated contouring wasused to define a volume of interest (VOI), comprising secondarytrabecular bone dorsal to the proximal femur growth plate and extending1.5 mm distal to primary trabecular bone. The cortical bone was excludedby placement of the VOI boundaries within the inner boundary of thecortical bone. Prior to image segmentation, a constrainedthree-dimensional (3D) Gaussian low-pass filter was applied to the imagedata for noise suppression (filter sigma=0.5, filter support=1). Aglobal threshold (0.36 gHA/cm3) was applied to extract a “binarized”trabecular structure from the VOL. The trabecular segmentation thresholdwas chosen by visual inspection of the segmentation results from arepresentative subset of the samples. The trabecular structuralcharacteristics were quantified by direct 3D morphometric analysis.Previous studies have shown that morphometric analysis of trabecularbone by microcomputed tomography is well correlated with similarestimates made by histomorphometry.

As shown in FIG. 28B, chronic BsAb20 treatment of DIO mice for 6 weeksresulted in the expected changes in metabolic parameters without anynegative signal in various bone parameters in tibial trabecular andfemoral cortical bones based on micro-computed tomography.

As shown in FIG. 29 , injection of BsAb17 into DIO mice did not increaseserum corticosterone levels above control. Chronic positive energybalance common in the modem society has been driving the obesitypandemic and the associated metabolic derangements characterized byinsulin resistance, hyperinsulinemia, glucose intolerance,hyperlipidemia, and hepatosteatosis, which often lead to severeillnesses such as type 2 diabetes, cirrhosis, stroke and heart disease.In 2009, the presence of UCP1-positive BAT in adult humans and theirfunctional ignificance in driving EE via heat dissipation were reported,igniting an immense interest in therapeutic induction and activation ofBAT for the treatment of obesity and related metabolic disease(Yoneshiro and Saito, Ann. Med., 1-9 (2014)).

However, most known BAT-activating mechanisms also induce white adiposetissue lipolysis, which may have a negative impact on cardiovascularoutcome (Dong et al., Cell Metab. 18, 118-129 (2013)). Of note, BATtransplant increases EE and induces weight loss without change in RQ(Stanford et al., J. Clin. Invest. 123, 215-223 (2013)). In this regard,FGF21 and anti-FGFR1/KLB agonist antibody described herein present aunique approach to selectively induce thermogenic response in BATwithout changing RQ, thus mimicking BAT transplant, rather thannon-specific sympathoactivation. In addition, based on what was observedin mice, it is envisioned that antibody-mediated activation ofFGFR1c/KLB complex may provide a safer and more efficient mean foranti-obese and anti-diabetic therapy, as opposed to the broader FGFR/KLBcomplex activation by FGF21 or FGF19 analogs.

Example 10: Humanization of Anti-KLB Antibody 8C5

The murine light chain CDRs of 8C5 were grafted into the human Kappa2and Kappa4 light chain frameworks. In addition to the primary graft,point mutations were also generated in each such that position 4 of thelight chain was converted to a leucine (designated “M4L”). Analysis wasperformed to identify those that expressed the best and did not showsignificant aggregation. Similarly, the heavy chain CDRs were graftedinto the human H1, H2, H3 and H4 IgG1 heavy chain frameworks. Variousresidues in the heavy chain backbones were mutated as follows: for H1the following changes were introduced: K71R, N73T and V78A (parentdesignated as “KNV” and construct designated “RTA”); for H2 thefollowing change was introduced: N73T (parent designated as “KNV” andconstruct designated “KTV”); for H3 the following changes wereintroduced: K71R and V78L (parent designated as “KNV” and constructdesignated “RNL”; and for H4 the following changes were introduced:K71V, N73T, and V78F (parent designated as “KNV” and constructdesignated “VTF”).

Antibodies based on all pairwise combinations of the 4 light chains and8 heavy chains (for a total of 32 antibodies) were produced andexpression levels and affinity were tested. Based on these experiments,the 8C5 derived light chain K4.M4L and the heavy chain H3.KNV exhibitedthe best combination of expression level and desired affinity.

The sequences of the 8C5.K4.M4L.H3.KNV variable regions and full-lengthantibody are as follows:

8C5.K4.M4L.H3.KNV Heavy Chain Variable Region (SEQ ID NO: 128)EVQLVESGGGLVQPGGSLRLSCAASDFSLTTYGVHWVRQAPGKGLEWLGVIWSGGSTDYNAAFISRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARD YGSTYVDAIDYWGQGTLVTVSS8C5.K4.M4L.H3.KNV Full Heavy Chain (SEQ ID NO: 129)EVQLVESGGGLVQPGGSLRLSCAASDFSLTTYGVHWVRQAPGKGLEWLGVIWSGGSTDYNAAFISRLTISKDNSKNTVYLQMNSLRAEDTAVYYCARDYGSTYVDAIDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK8C5.K4.M4L.H3.KNV Light Chain Variable Region (SEQ ID NO: 130)DIVLTQSPDSLAVSLGERATINCRASESVESYGNRYMTWYQQKPGQPPKLLIYRAANLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNED PWTFGQGTKVEIK8C5.K4.M4L.H3.KNV Full Light Chain (SEQ ID NO: 131)DIVLTQSPDSLAVSLGERATINCRASESVESYGNRYMTWYQQKPGQPPKLLIYRAANLQSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

Example 11: Generation of an Anti-FGFR1 Antibody Hybrid Between YW182.3and YW182.5

YW182.5, which was the anti-FGFR1 arm that did not activate theKLB/FGFR1c complex in the absence of an anti-FGFR1 arm, was discoveredto give good results when combined with 8C5, and has a tryptophan asposition 33 of the heavy chain which is susceptible to oxidation.YW182.2, which appears to bind the same epitope as YW182.5, also hassuch a tryptophan at position 33 of the heavy chain. Several mutationswere introduced at this position to obviate this problem: for YW182.5W33Y, W33H, W33F and W33L were introduced and for YW182.2 W33Y and W33Fwere introduced. Surprisingly, the introduced mutations had differenteffects in the two antibodies. In the case of YW182.2, it was observedthat the mutations did not appreciably affect the affinity or agonisticactivity for FGFR1, whereas for YW182.5 the mutations greatly decreasedthe affinity and agonistic activity for FGFR1 (see, for example, FIG. 31). Therefore, experiments were performed to identify an antibody withthe W33Y mutations, but with an affinity closer to that of the YW182.5antibody using two approaches.

In one approach, for the YW182.2 W33Y heavy chain sequence alaninescanning across CDR3 was performed, mutating positions 95, 96, 97, 98,99, 100, 100a and 100b to alanine. The affinity of the resultingantibodies were analyzed and those that retained the very high affinityof the YW182.2 W33Y parent were identified (Table 9).

TABLE 9 Affinity of YW182.2 derivatives. Antibody EC₅₀ (nM)YW182.2_W33Y_96A 2.4 YW182.2_W33Y_97A 5.3 YW182.2_W33Y_100A 5.8YW182.2_W33Y_98A 8.8 YW182.2_W33Y_GDY 11.1 YW182.5 34.6YW182.2_W33Y_100aA 55.1 YW182.2_W33Y_95A 221.1 YW182.2_W33Y_99A 316.2YW182.2_W33Y_100bA None detected

In a second approach, CDRs from the YW182.2 W33Y antibody (with veryhigh affinity) and the YW182.5 W33Y antibody (with almost no binding)were mix-and-matched. The YW182.2 W33Y and YW182.5 W33Y antibodies haveidentical CDR sequences in the light chain (CDR-L1, RASQDVSTAVA (SEQ IDNO: 139); CDR-L2, SASFLYS (SEQ ID NO: 140); and CDR-L3 QQSYTTPPT (SEQ IDNO: 141) a single amino acid difference in CDR-H1 (YW182.2 W33Y CDR-H1,STYIS (SEQ ID NO: 152) and YW182.5 W33Y CDR-H1, SNYIS (SEQ ID NO: 136));three amino acid differences in or adjacent to CDR-H2 (YW182.2 W33YCDR-H2, EIDPYDGDTYYADSVKG (SEQ ID NO: 137 and YW182.5 W33Y,EIDPYDGATDYADSVKG (SEQ ID NO: 153)); and very difference CDR-H3sequences (YW182.2 W33Y, EHFDAWVHYYVMDY (SEQ ID NO: 154) and YW182.5W33Y GTDVMDY (SEQ ID NO: 138). Antibodies with heavy chains based on allpossible combinations of heavy chain CDRs from YW182.5 W33Y and YW182.2W33Y (eight including the two parental antibodies) were constructed andtested. Most of the antibodies had affinity similar to one or the other,but, surprisingly, one combination demonstrated binding that was nearlyidentical to the parent YW182.5 antibody. This antibody has the CDR-H1and CDR-H3 from YW182.5 W33Y, but the CDR-H2 from YW182.2 W33Y. Thisantibody was designated as “YW182.5 YGDY” to represent the followingchanges in the YW182.5 sequence: W33Y, A49G, A56D, and D58Y.

The sequences of the YW182.5 YGDY antibody are as follows:

YW182.5 YGDY Heavy Chain Variable Region (SEQ ID NO: 132)EVQLVESGGGLVQPGGSLRLSCAASGFTFTSNYISWVRQAPGKGLEWVGEIDPYDGDTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAT GTDVMDYWGQGTLVTVSS.YW182.5 YGDY Full Heavy Chain (SEQ ID NO: 133)EVQLVESGGGLVQPGGSLRLSCAASGFTFTSNYISWVRQAPGKGLEWVGEIDPYDGDTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCATGTDVMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPGK.YW182.5 YGDY Light Chain Variable Region (SEQ ID NO: 134)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTF GQGTKVEIK.YW182.5 YGDY Full Light Chain (SEQ ID NO: 135)DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC

Example 12: Testing of Bispecific Antibodies with Humanized 8C5 andAnti-FGFR1 Variants

Various bispecific antibody combinations of 8C5.K4H3.M4L.KNV anddifferent anti-FGFR1 arms were tested in the GAL-ELK1-based luciferaseassay in HEK293 cells expressing FGFR1c with or without KLB. Aspreviously observed, each bispecific antibody combination inducedluciferase activity in a dose-dependent manner in cells expressingrecombinant hFGFR1c and hKLB, but not in cells without KLB expression(FIG. 30 ). These data confirm that these modified variants retain theadvantages of the parent antibodies, e.g., BsAb13. The binding affinityof an anti-KLB/anti-FGFR1 antibody that has a humanized 8C5 arm(8C5.K4.M4L.H3.KNV) and a YW182.5_YGDY arm to human, cynomolgus monkeyand mouse KLB/FGFR1c complexes on the surface of HEK293 cells are shownin Table 10.

TABLE 10 Binding affinities. anti-KLB/anti-FGFR1c Average Standard CellLine antibody K_(d) (nM) K_(d) (nM) deviation 293huKLB/huR1c 1.87 1.880.06 1.95 1.83 293cynoKLB/cynoR1c 2.54 2.55 0.25 2.80 2.31293msKLB/msR1c 4.12 3.92 0.17 3.85 3.80

In addition to the various embodiments depicted and claimed, thedisclosed subject matter is also directed to other embodiments havingother combinations of the features disclosed and claimed herein. Assuch, the particular features presented herein can be combined with eachother in other manners within the scope of the disclosed subject mattersuch that the disclosed subject matter includes any suitable combinationof the features disclosed herein. The foregoing description of specificembodiments of the disclosed subject matter has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the disclosed subject matter to those embodimentsdisclosed.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions and methodsof the disclosed subject matter without departing from the spirit orscope of the disclosed subject matter. Thus, it is intended that thedisclosed subject matter include modifications and variations that arewithin the scope of the appended claims and their equivalents.

Various publications, patents and patent applications are cited herein,the contents of which are hereby incorporated by reference in theirentireties.

What is claimed is:
 1. A composition comprising: (a) an isolatedbispecific antibody, or an antigen-binding portion thereof, that bindsto beta-Klotho (KLB) and Fibroblast Growth Factor Receptor 1 (FGFR1);(b) a nucleic acid encoding a bispecific antibody, or an antigen-bindingportion thereof, that binds to beta-Klotho (KLB) and Fibroblast GrowthFactor Receptor 1 (FGFR1); (c) a host cell comprising a nucleic acidencoding a bispecific antibody, or an antigen-binding portion thereof,that binds to beta-Klotho (KLB) and Fibroblast Growth Factor Receptor 1(FGFR1); (d) an anti-KLB antibody, or an antigen-binding portionthereof, or (e) a pharmaceutical formulation comprising an isolatedbispecific antibody, or an antigen-binding portion thereof, that bindsto beta-Klotho (KLB) and Fibroblast Growth Factor Receptor 1 (FGFR1). 2.The composition of claim 1, wherein the bispecific antibody, or anantigen-binding portion thereof, binds to an FGFR1 epitope within afragment of FGFR1c comprising the amino acid sequence set forth in SEQID NO: 143 or SEQ ID NO:
 144. 3. The composition of claim 1, wherein thebispecific antibody, or an antigen-binding portion thereof, binds to aKLB epitope within a fragment of KLB consisting of the amino acidsequence (SEQ ID NO: 142) SSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS.


4. The composition of claim 1, wherein the bispecific antibody, or anantigen-binding portion thereof, comprises (a) a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 128 and (b) alight chain variable region comprising the amino acid sequence of SEQ IDNO:
 130. 5. The composition of claim 1, wherein the bispecific antibody,or an antigen-binding portion thereof, comprises (a) a heavy chaincomprising the amino acid sequence of SEQ ID NO: 132 and (b) a lightchain comprising the amino acid sequence of SEQ ID NO:
 134. 6. Thecomposition of claim 1, wherein the bispecific antibody, or anantigen-binding portion thereof, comprises: (a) a heavy chain variableregion CDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-15, and conservative substitutions thereof;(b) a heavy chain variable region CDR2 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 16-31, andconservative substitutions thereof; (c) a heavy chain variable regionCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 32-47, and conservative substitutions thereof;(d) a light chain variable region CDR1 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 48-62, andconservative substitutions thereof; (e) a light chain variable regionCDR2 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 63-78, and conservative substitutions thereof;and (f) a light chain variable region CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 79-93,and conservative substitutions thereof.
 7. The composition of claim 1,wherein the bispecific antibody, or an antigen-binding portion thereof,comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:136, (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 137,(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 138, (d)HVR-L1 comprising the amino acid sequence of SEQ ID NO: 139, (e) HVR-L2comprising the amino acid sequence of SEQ ID NO: 140, and (f) HVR-L3comprising the amino acid sequence of SEQ ID NO:
 141. 8. The compositionof claim 1, wherein the anti-KLB antibody, or an antigen-binding portionthereof, comprises: (a) a heavy chain variable region CDR1 comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:1-15, and conservative substitutions thereof; (b) a heavy chain variableregion CDR2 domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16-31, and conservative substitutionsthereof; and (c) a heavy chain variable region CDR3 domain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:32-47, and conservative substitutions thereof.
 9. The composition ofclaim 1, wherein the anti-KLB antibody, or an antigen-binding portionthereof, comprises: (a) a light chain variable region CDR1 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 48-62, and conservative substitutions thereof; (b) a lightchain variable region CDR2 domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 63-78, andconservative substitutions thereof; and (c) a light chain variableregion CDR3 domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 79-93, and conservative substitutionsthereof.
 10. The composition of claim 1, wherein the pharmaceuticalformulation further comprises a pharmaceutically acceptable carrier. 11.The composition of claim 1, wherein the nucleic acid encodes abispecific antibody, or an antigen-binding portion thereof, that bindsto beta-Klotho (KLB) and Fibroblast Growth Factor Receptor 1 (FGFR1).12. The composition of claim 1, wherein the host cell comprises anucleic acid encoding a bispecific antibody, or an antigen-bindingportion thereof, that binds to beta-Klotho (KLB) and Fibroblast GrowthFactor Receptor 1 (FGFR1).
 13. A method of treating an individual havinga disease selected from the group consisting of polycystic ovarysyndrome (PCOS), metabolic syndrome (MetS), obesity, non-alcoholicsteatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD),hyperlipidemia, hypertension, type 2 diabetes, non-type 2 diabetes, type1 diabetes, latent autoimmune diabetes (LAD), maturity onset diabetes ofthe young (MODY), and aging and related diseases such as Alzheimer'sdisease, Parkinson's disease and ALS, Bardet-Biedl syndrome,Prader-Willi syndrome, Alstrom syndrome, Cohen syndrome, Albright'shereditary osteodystrophy (pseudohypoparathyroidism), Carpentersyndrome, MOMO syndrome, Rubinstein-Taybi syndrome, fragile X syndromeand Börjeson-Forssman-Lehman syndrome, comprising administering to theindividual an effective amount of one or more antibodies, wherein theone or more antibodies is selected from the group consisting of anisolated bispecific antibody, or an antigen-binding portion thereof,that binds to beta-Klotho (KLB) and Fibroblast Growth Factor Receptor 1(FGFR1), and an anti-KLB antibody, or an antigen-binding portionthereof.
 14. The method of claim 13, wherein the one or more antibodiesreduces blood glucose levels in vivo.
 15. The method of claim 13,wherein the bispecific antibody, or an antigen-binding portion thereof,to a KLB epitope within a fragment of KLB consisting of the amino acidsequence (SEQ ID NO: 142) SSPTRLAVIPWGVRKLLRWVRRNYGDMDIYITAS.


16. The method of claim 13, wherein the bispecific antibody, or anantigen-binding portion thereof, comprises: (a) a heavy chain variableregion CDR1 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-15, and conservative substitutions thereof;(b) a heavy chain variable region CDR2 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 16-31, andconservative substitutions thereof; (c) a heavy chain variable regionCDR3 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 32-47, and conservative substitutions thereof;(d) a light chain variable region CDR1 domain comprising an amino acidsequence selected from the group consisting of SEQ ID NOs: 48-62, andconservative substitutions thereof; (e) a light chain variable regionCDR2 domain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 63-78, and conservative substitutions thereof;and (f) a light chain variable region CDR3 domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 79-93,and conservative substitutions thereof.
 17. The composition of claim 13,wherein the anti-KLB antibody, or an antigen-binding portion thereof,comprises: (a) a heavy chain variable region CDR1 comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 1-15,and conservative substitutions thereof; (b) a heavy chain variableregion CDR2 domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 16-31, and conservative substitutionsthereof; and (c) a heavy chain variable region CDR3 domain comprising anamino acid sequence selected from the group consisting of SEQ ID NOs:32-47, and conservative substitutions thereof.
 18. The method of claim13, wherein the anti-KLB antibody, or an antigen-binding portionthereof, comprises: (a) a light chain variable region CDR1 domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 48-62, and conservative substitutions thereof; (b) a lightchain variable region CDR2 domain comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 63-78, andconservative substitutions thereof; and (c) a light chain variableregion CDR3 domain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 79-93, and conservative substitutionsthereof.
 19. A method of producing an antibody comprising culturing acell host cell that comprises a nucleic acid encoding a bispecificantibody, or an antigen-binding portion thereof, that binds tobeta-Klotho (KLB) and Fibroblast Growth Factor Receptor 1 (FGFR1). 20.The method of claim 19, wherein the bispecific antibody, or anantigen-binding portion thereof, comprises (a) a first antibody, orantigen binding portion thereof, comprising a heavy chain variableregion and a light chain variable region, wherein the heavy chainvariable region comprises amino acids having a sequence that is at least95% identical to the sequence set forth in SEQ ID NO: 128, and the lightchain variable region comprises amino acids having a sequence that is atleast 95% identical to the sequence set forth in SEQ ID NO: 130; and (b)a second antibody, or antigen binding portion thereof, comprising aheavy chain variable region and a light chain variable region, whereinthe heavy chain variable region comprises amino acids having a sequencethat is at least 95% identical to the sequence set forth in SEQ ID NO:132, and the light chain variable region comprises amino acids having asequence that is at least 95% identical to the sequence set forth in SEQID NO: 134.